Mammalian genes; related reagents and methods

ABSTRACT

Purified genes encoding cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this molecule are provided. Methods of using said reagents and diagnostic kits are also provided.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/164,616, filed Nov. 10, 1999, and Provisional PatentApplication No. 60/153,281, filed Sep. 9, 1999, each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to compositions and methods related toproteins which function in controlling biology and physiology ofmammalian cells, e.g., cells of a mammalian immune system. Inparticular, it provides purified genes, proteins, antibodies, relatedreagents, and methods useful, e.g., to regulate activation, development,differentiation, and function of various cell types, includinghematopoietic cells.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. See, e.g., Paul, (1998) Fundamental Immunology (4th ed.) RavenPress, N.Y. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders. Some of thesefactors are hematopoietic growth factors, e.g., granulocyte colonystimulating factor (G-CSF). See, e.g., Thomson, (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Mire-Sluis and Thorpe, (ed.1998) Cytokines Academic Press, San Diego; Metcalf and Nicola, (1995)The Hematopoietic Colony Stimulating Factors Cambridge University Press;and Aggarwal and Gutterman, (1991) Human Cytokines Blackwell Pub.Cytokine expression by cells of the immune system plays an importantrole in the regulation of the immune response. Most cytokines arepleiotropic and have multiple biological activities, includingantigen-presentation; activation; proliferation and differentiation ofCD4+ T cell subsets; antibody response by B cells; and manifestations ofhypersensitivity. In addition cytokines may be used in the diagnosis andtherapy of a wide range of degenerative or abnormal conditions whichdirectly or indirectly involve the immune system and/or hematopoieticcells.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, and/ordifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

From the foregoing, it is evident that the discovery and development ofnew lymphokines, e.g., related to G-CSF and/or IL-6, could contribute tonew therapies for a wide range of degenerative or abnormal conditionswhich directly or indirectly involve the immune system and/orhematopoietic cells. In particular, the discovery and development oflymphokines which enhance or potentiate the beneficial activities ofknown lymphokines would be highly advantageous. Originally the novelgene IL-B30 was identified as a potential cytokine based on itspredicted structure and was classified as a long-chain cytokine likeIL-6 and G-CSF (International Patent Application PCT/US98/15423 (WO99/05280). IL-6 and related cytokines like Oncostatin M, leukemiainhibitory factor (LIF), ciliary neurotrophic factor (CNTF) andcardiothrophin-1 have biological activities on hematopoiesis,thrombopoiesis, induction of an acute phase response, osteoclastformation, neuron differentiation and survival, and cardiac hypertrophy.Transgenic expression of IL-B30 in mice induced a similar phenotype asthat observed after overexpression of IL-6 in mice, comprising runting,systemic inflammation, infertility and death. IL-B30 appears to be anovel cytokine involved in inflammation.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of thephysiological role of IL-B30, also referred to herein as the IL-B30protein, and its role in the immune response. In particular, the role ofIL-B30 has been elucidated in pathways involved in inflammation,infectious disease, hematopoietic development, and viral infection. Theinvention is specifically directed to compositions comprisingcombinations of IL-12 p40 subunit with interleukin-B30 (IL-B30) andtheir biological activities. It includes nucleic acids coding for bothpolypeptides or fusion proteins, and methods for their production anduse. The nucleic acids of the invention are characterized, in part, bytheir homology to complementary DNA (cDNA) sequences disclosed herein,and/or by functional assays. Also provided are polypeptides, antibodies,and methods of using them, including using nucleic acid expressionmethods. Methods for modulating or intervening in the control of agrowth factor dependent physiology or an immune response are provided.

The present invention is based, in part, upon the discovery that the p40subunit of IL-12 also associates with the IL-B30 cytokine, describedpreviously, e.g., in U.S. Ser. Nos. 08/900,905 and 09/122,443, in anatural form. Thus, the coexpression of the two polypeptides togetherresults in functional receptor binding and signaling.

The present invention provides compositions comprising: a) both asubstantially pure polypeptide comprising a plurality of distinctsegments of at least 7 contiguous amino acid from IL-12 p40 and asubstantially pure polypeptide comprising a plurality of distinctsegments of at least 7 contiguous amino acids from IL-B30; b) both asubstantially pure polypeptide comprising at least 11 contiguous aminoacids from IL-12 p40 and a substantially pure polypeptide comprising atleast 11 contiguous amino acids from IL-B30; c) a substantially purepolypeptide comprising both a plurality of distinct segments of at least7 contiguous amino acids of IL-12 p40 and a plurality of distinctsegments of at least 7 contiguous amino acids of IL-B30; or d) asubstantially pure polypeptide comprising both a segment of at least 11contiguous amino acids of IL-12 p40 and a segment of at least 11contiguous amino acids of IL-B30. Various embodiments include suchcompositions: a) wherein the described plurality of distinct segments ofat least 7 contiguous amino acids comprise one segment of at least 9contiguous amino acids; b) wherein the described plurality of distinctsegments of at least 7 contiguous amino acids are both at least 9contiguous amino acids; c) wherein the described segment of at least 11contiguous amino acids of IL-12 p40 is at least 15 contiguous aminoacids; d) wherein the described segment of at least 11 contiguous aminoacids of IL-B30 is at least 15 contiguous amino acids; e) furthercomprising a carrier selected from an aqueous compound, including water,saline, and/or buffer; f) formulated for oral, rectal, nasal, topical,or parenteral administration; or g) which is sterile composition. Otherembodiments include those: a) wherein at least one of the describedpolypeptides is: i) detectably labeled; ii) recombinantly produced; iii)unglycosylated; iv) denatured; v) attached to a solid substrate; or vi)conjugated to another chemical moiety; b) comprising both asubstantially pure IL-12 p40 polypeptide and a substantially pure IL-B30polypeptide; c) comprising a substantially pure polypeptide comprisingIL-12 p40 fused to IL-B30; or d) combined with IL-18, IL-12, radiationor chemotherapy, an immune adjuvant, or an anti-viral. Kit embodimentsinclude those comprising such a described composition and: a) acompartment comprising the described polypeptide; or b) instructions foruse or disposal of reagents in the described kit.

Nucleic acid compositions of the invention include, e.g., an isolated orrecombinant nucleic acid encoding: a) both a substantially purepolypeptide comprising a plurality of distinct segments of at least 7contiguous amino acid from IL-12 p40 and a substantially purepolypeptide comprising a plurality of distinct segments of at least 7contiguous amino acids from IL-B30; b) both a substantially purepolypeptide comprising at least 11 contiguous amino acids from IL-12 p40and a substantially pure polypeptide comprising at least 11 contiguousamino acids from IL-B30; c) a substantially pure polypeptide comprisingboth a plurality of distinct segments of at least 7 contiguous aminoacids of IL-12 p40 and a plurality of distinct segments of at least 7contiguous amino acids of IL-B30; or d) a substantially pure polypeptidecomprising both a segment of at least 11 contiguous amino acids of IL-12p40 and a segment of at least 11 contiguous amino acids of IL-B30.Various embodiments include such a nucleic acid: a) wherein thedescribed plurality of distinct segments of at least 7 contiguous aminoacids comprise one segment of at least 9 contiguous amino acids; b)wherein the described plurality of distinct segments of at least 7contiguous amino acids are both at least 9 contiguous amino acids; c)wherein the described segment of at least 11 contiguous amino acids ofIL-12 p40 is at least 15 contiguous amino acids; d) wherein thedescribed segment of at least 11 contiguous amino acids of IL-B30 is atleast 15 contiguous amino acids; e) wherein the described IL-12 p40 isfrom a primate; f) wherein the described IL-B30 is from a primate; g)which is an expression vector; h) which further comprises an origin ofreplication; i) which comprises a detectable label; j) which comprisessynthetic nucleotide sequence; k) which is less than 6 kb, preferablyless than 3 kb; or l) which is from primate. Also provided is a cellcomprising the described recombinant nucleic acid, including wherein thedescribed cell is: a prokaryotic, eukaryotic, bacterial, yeast, insect,mammalian, mouse, primate, or human cell. Kit embodiments include thosecomprising a described nucleic acid and: a) a compartment comprising thedescribed nucleic acid; b) a compartment further comprising a primateIL-12 p40 polypeptide; c) a compartment further comprising a primateIL-B30 polypeptide; or d) instructions for use or disposal of reagentsin the described kit.

Alternatively, the invention provides a nucleic acid which hybridizes:a) under wash conditions of 30 minutes at 50° C. and less than 1M saltto the natural mature coding portion of primate IL-12 p40; and b) underwash conditions of 30 minutes at 50° C. and less than 1M salt to thenatural mature coding portion of primate IL-B30. Various embodimentsinclude such a described nucleic acid wherein: a) the described washconditions for IL-12 p40 are at 60° C. and less than 400 mM salt; b) thedescribed wash conditions for IL-B30 are at 60° C. and less than 400 mMsalt; c) the described nucleic acid exhibits identity over a stretch ofat least 50 nucleotides to sequence encoding primate IL-12 p40; and/ord) the described nucleic acid exhibits identity over a stretch of atleast 50 nucleotides to sequence encoding primate IL-B30. Preferredembodiments include such a nucleic acid wherein: a) the described washconditions for IL-12 p40 are at 65° C. and less than 150 mM salt; b) thedescribed wash conditions for IL-B30 are at 65° C. and less than 150 mMsalt; c) the described nucleic acid exhibits identity over a stretch ofat least 90 nucleotides to sequence encoding primate IL-12 p40; and/ord) the described nucleic acid exhibits identity over a stretch of atleast 90 nucleotides to sequence encoding primate IL-B30.

Antagonists of the IL-12 p40/IL-B30 compositions are provided, combinedwith, e.g., a TNFα antagonist, an IL-12 antagonist, IL-10, or steroids.

The invention also provides a binding compound, e.g., comprising anantigen binding site from an antibody, which antibody specifically bindsto an IL-12 p40/IL-B30 composition, as described, a) comprising asubstantially pure polypeptide comprising both a substantially pureIL-12 p40 polypeptide and a substantially pure IL-B30 polypeptide; or b)comprising a substantially pure polypeptide comprising IL-12 p40 fusedto IL-B30; but not to either IL-12 p40 or IL-B30 polypeptide. Otherbinding compounds include those wherein: a) the described bindingcompound is in a container; b) the described binding compound is an Fv,Fab, or Fab2 fragment; c) the described binding compound is conjugatedto another chemical moiety; or d) the described antibody: i) is raisedagainst an IL-12 p40/IL-B30 composition; ii) is immunoselected; iii) isa polyclonal antibody; iv) exhibits a Kd to antigen of at least 30 mM;v) is attached to a solid substrate, including a bead or plasticmembrane; vi) is in a sterile composition; or vii) is detectablylabeled, including a radioactive or fluorescent label. Certain preferredforms include compositions comprising: a) a sterile binding compound, asdescribed; or b) the described binding compound and a carrier, whereinthe described carrier is: i) an aqueous compound, including water,saline, and/or buffer; and/or ii) formulated for oral, rectal, nasal,topical, or parenteral administration. Additionally, kit embodiments areprovided comprising the described binding compound and: a) a compartmentcomprising the described binding compound; or b) instructions for use ordisposal of reagents in the described kit.

Moreover, the invention provides methods for producing anantigen:antibody complex, comprising contacting, under appropriateconditions, a primate IL-12 p40/IL-B30 composition with a describedbinding compound, thereby allowing the described complex to form.Various methods include those wherein: a) the described complex ispurified from other cytokines; b) the described complex is purified fromother antibody; c) the described contacting is with a sample comprisinga cytokine; d) the described contacting allows quantitative detection ofthe described antigen; e) the described contacting is with a samplecomprising the described antibody; or f) the described contacting allowsquantitative detection of the described antibody.

The invention also provides methods of modulating physiology ordevelopment of a cell or tissue comprising contacting the described cellwith an IL-12 p40/IL-B30 composition, or antagonist thereof. Onepreferred method is modulating physiology or development of a cellcomprising contacting the described cell with an IL-12 p40/IL-B30composition, and the described contacting results in an increase inproduction of IFNγ. Typically, the described cell is in a host organism,and the described organism exhibits an enhanced Th1 response, e.g., oneselected from an: anti-tumor effect; adjuvant effect; anti-viral effect;or antagonized allergic effect. Often, the contacting is in combinationwith: IL-18; IL-12; radiation therapy or chemotherapy; an immuneadjuvant; or an anti-viral therapeutic.

In another embodiment, the described antagonist is an antibody againstIL-12 receptor subunit β1. Thus, the invention also embraces a method,as described, wherein the described contacting is with an antagonist,and the described contacting results in a relative decrease inproduction of IFNγ. Thus, the invention provides methods of modulatingphysiology or development of a cell in a host organism, comprisingadministering the described antagonist to the described organism,wherein the described contacting results in amelioration of: anautoimmune condition or a chronic inflammatory condition.

The identification of the association of the two subunits providesmethods of increasing the secretion of: a) a primate IL-B30, such methodcomprising expressing the described polypeptide with IL-12 p40; or b) aprimate IL-12 p40, such method comprising expressing the described IL-12p40 with IL-B30. Preferably, either: a) the described increasing is atleast 3-fold; or b) the described expressing is of a recombinant nucleicacid encoding IL-B30 and IL-12 p40.

Methods for screening for a receptor which binds the described IL-12p40/IL-B30 composition are provided, e.g., comprising contacting thedescribed complex to a cell expressing the described receptor underconditions allowing the described complex to bind to the describedreceptor, thereby forming a detectable interaction. Preferably, thedescribed interaction results in a physiological response in thedescribed cell.

The present invention also provides methods of modulating thetrafficking or activation of a leukocyte in an animal, the methodscomprising contacting monocyte/macrophage lineage cells in the animalwith a therapeutic amount of an agonist of a mammalian IL-B30 protein;or an antagonist of a mammalian IL-B30 protein. Preferred embodimentsinclude where: the mammalian IL-B30 protein is a primate protein; and/orthe antagonist is an antibody which binds to the mammalian IL-B30.Certain embodiments include where the monocyte/macrophage lineage cellsinclude a microglial cell or a dendritic cell, or where the animalexhibits signs or symptoms of an inflammatory, leukoproliferative,neurodegenerative, or post-traumatic condition. Preferred embodimentsinclude where the sign or symptom is in lung tissue; liver tissue;neural tissue; lymphoid tissue; myeloid tissue; pancreas;gastrointestinal tissue; thyroid tissue; muscle tissue; or skin orcollagenous tissue.

Other methods include where the modulating is inhibiting function of theleukocyte cell; and/or where the administering is the agonist.Preferably, the agonist is the mammalian IL-B30.

Certain embodiments include where the animal is experiencing signs orsymptoms of autoimmunity; an inflammatory condition; tissue specificautoimmunity; degenerative autoimmunity; rheumatoid arthritis;osteoarthritis; atherosclerosis; multiple sclerosis; vasculitis; delayedhypersensitivities; skin grafting; a transplant; spinal injury; stroke;neurodegeneration; an infectious disease; ischemia; cancer; tumors;multiple myeloma; Castleman's disease; postmenopausal osteoporosis orIL-6-associated diseases. The administering may be in combination with:an anti-inflammatory cytokine agonist or antagonist; an analgesic; ananti-inflammatory agent; or a steroid.

Various other methods are provided where the modulating is enhancingfunction of the leukocyte cell, and/or the administering is theantagonist. Preferably, the antagonist is: an antibody which binds tothe mammalian IL-B30; or a mutein of the mammalian IL-B30 which competeswith the mammalian IL-B30 in binding to an IL-B30 receptor, but does notsubstantially signal. In various embodiments, the method is appliedwhere the animal experiences signs or symptoms of wound healing or clotformation. The administering will often be in combination with: anangiogenic factor; a growth factor, including FGF or PDGF; anantibiotic; or a clotting factor.

Lastly, the present invention provides a method of inducing theproliferation of memory T-cells by administering IL-B30 or an agonistthereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

OUTLINE

-   -   I. General    -   II. Purified IL-12 p40/IL-B30 complex        -   A. physical properties        -   B. biological properties    -   III. Physical Variants        -   A. sequence variants, fragments        -   B. post-translational variants            -   1. glycosylation            -   2. others    -   IV. Functional Variants        -   A. analogs, fragments            -   1. agonists            -   2. antagonists        -   B. mimetics            -   1. protein            -   2. chemicals        -   C. species variants    -   V. Antibodies        -   A. polyclonal        -   B. monoclonal        -   C. fragments, binding compositions    -   VI. Nucleic Acids        -   A. natural isolates; methods        -   B. synthetic genes        -   C. methods to isolate    -   VII. Making p40/IL-B30 complex, mimetics        -   A. recombinant methods        -   B. synthetic methods        -   C. natural purification    -   VIII. Uses        -   A. diagnostic        -   B. therapeutic    -   IX. Kits        -   A. nucleic acid reagents        -   B. protein reagents        -   C. antibody reagents    -   X. Isolating receptors for p40/IL-B30 complexes        I. General

The present invention provides description and teaching of pairing ofmammalian proteins to make a soluble cytokine, e.g., a secreted moleculewhich can mediate a signal between immune or other cells. See, e.g.,Paul, (1998) Fundamental Immunology (4th ed.) Raven Press, N.Y. Certainsoluble factors are made up of heterodimer polypeptides, e.g., IL-6 andIL-12. The dimer forms, which are likely the physiological forms, andfragments, or antagonists will be useful, e.g., in physiologicalmodulation of cells expressing a receptor. It is likely that thefunctional cytokine comprising p40/IL-B30 complex has either stimulatoryor inhibitory effects on hematopoietic cells, including, e.g., lymphoidcells, such as T-cells, B-cells, natural killer (NK) cells, macrophages,dendritic cells, hematopoietic progenitors, etc. The proteins will alsobe useful as antigens, e.g., immunogens, for raising antibodies tovarious epitopes on the protein, both linear and conformationalepitopes.

The LL-12 p4.0 subunit has been described. See, e.g., Seiler et al.,U.S. Pat. No. 5,547,852; Scott and Trinchieri, U.S. Pat. No. 5,571,515;Gately et al., U.S. Pat. No. 5,650,492 (disclosing SEQ ID NO:6);Liesehke and Mulligan, U.S. Pat. No. 5,891,680; Warne et al., U.S. Pat.No. 5,744,132; and accession numbers gbM86671 (SEQ ID NO:7; encoding SEQID NO:8; from Mus musculus), gbAF133197 (SEQ ID NO:9; encoding SEQ IDNO:10; from Rattus norvegicus), gbU16674 (SEQ ID NO:11; encoding SEQ IDNO:12; from Rattus norvegicus), gbU83184 (SEQ ID NO:13; encoding SEQ IDNO:14; from Felis catus), embY07762 (SEQ ID NO:15; encoding SEQ IDNO:16; from Felis catus), embY11129.1 (SEQ ID NO:17; encoding SEQ IDNO:18; from Equus caballus), gbM65272 (SEQ ID NO:19; encoding SEQ IDNO:20; from human), gbAF007576 (SEQ ID NO:21; encoding SEQ ID NO:22;from Capra hircus), gbU19841 SE ID NO:23; encoding SEQ ID NO:24; fromMacaca mulatta),gbU11815 (SEQ ID NO:25; encoding SEQ ID NO:26; from Bostaurus), gbU57752 (SEQ ID NO:27; encoding SEQ ID NO:28; from Cervuselaphus), gbAF004024 (SEQ ID NO:29; encoding SEQ ID NO:30; from Ovisaries), gbU49100 (SEQ ID NO:31; encoding SEQ ID NO:32; from Canisfamiliaris), gbU19834 (SEQ ID NO:33; encoding SEQ ID NO:34; fromCercocebus torguatus), and embX97019 (SEQ ID NO:35; encoding SEQ IDNO:36; from M. monax). A sequence encoding IL-B30 was identified from ahuman genomic sequence. The molecule was designated huIL-B30. A rodentsequence, e.g., from mouse, was also described. See, e.g., U.S. Ser.Nos. 08/900,905 and 09/122,443. The present invention embracescompositions comprising combinations of these two polypeptides, e.g.,p40 and IL-B30, and nucleic acid constructs encoding both sequences.Antibodies which recognize the combinations are also provided, andmethods of producing the two messages or polypeptides, e.g.,coordinately.

The human IL-B30 gene encodes a small soluble cytokine-like protein, ofabout 198 amino acids. The psort predicted signal sequence probably isabout 17 residues, and would run from the Met to about Ala. See Table 1and SEQ. ID. NO: 1 and 2. IL-B30 exhibits structural motifscharacteristic of a member of the long chain cytokines. Compare, e.g.,IL-B30, G-CSF, and IL-6, sequences available from GenBank. See also U.S.Ser. Nos. 08/900,905 and 09/122,443.

TABLE 1 Nucleic acid (SEQ ID NO: 1) ending IL-B30 from a primate, e.g.,human. Translated amino acid sequence is SEQ ID NO:2. ATG CGT GGG AGCAGA GCT GTA ATG CTG CTG TTG CTG CTG CCC TGG ACA 48 Met Leu Gly Ser ArgAla Val Met Leu Leu Leu Leu Leu Pro Trp Thr−21 −20                 −15                 −10 GCT CAG GGC AGA GCT GTGCCT GGG GGC AGC AGC CCT GCC TGG ACT CAG 96 Ala Gln Gly Arg Ala Val ProGly Gly Ser Ser Pro Ala Trp Thr Gln −5                   1               5                  10 TGC CAG CAGCTT TCA CAG AAG CTC TGC ACA CTG GCC TGG AGT GCA CAT 144 Cys Gln Gln LeuSer Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His             15                  20                  25 CCA CTA GTG GGACAC ATG GAT CTA AGA GAA GAG GGA GAT GAA GAG ACT 192 Pro Leu Val Gly HisMet Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr         30                  35                  40 ACA AAT GAT GTT CCCCAT ATC CAG TGT GGA GAT GGC TGT GAC CCC CAA 240 Thr Asn Asp Val Pro HisIle Gln Cys Gly Asp Gly Cys Asp Pro Gln     45                  50                  55 GGA CTC AGG GAC AAC AGTCAG TTC TGC TTG CAA AGG ATC CAC CAG GGT 288 Gly Leu Arg Asp Asn Ser GlnPhe Cys Leu Gln Arg Ile His Gln Gly 60                  65                  70                  75 CTG ATTTTT TAT GAG AAG CTG CTA GGA TCG GAT ATT TTC ACA GGG GAG 336 Leu Ile PheTyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu                 80                  85                  90 CCT TCT CTGCTC CCT GAT AGC CCT GTG GCG CAG CTT CAT GCC TCC CTA 384 Pro Ser Leu LeuPro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu             95                 100                 105 CTG GGC CTC AGCCAA CTC CTG CAG CCT GAG GGT CAC CAC TGG GAG ACT 432 Leu Gly Leu Ser GlnLeu Leu Gln Pro Glu Gly His His Trp Glu Thr        110                 115                 120 CAG CAG ATT CCA AGCCTC AGT CCC AGC CAG CCA TGG CAG CGT CTC CTT 480 Gln Gln Ile Pro Ser LeuSer Pro Ser Gln Pro Trp Gln Arg Leu Leu    125                 130                 135 CTC CGC TTC AAA ATC CTTCGC AGC CTC CAG GCC TTT GTG GCT GTA GCC 528 Leu Arg Phe Lys Ile Leu ArgSer Leu Gln Ala Phe Val Ala Val Ala140                 145                 150                 155 GCC CGGGTC TTT GCC CAT GGA GCA GCA ACC CTG AGT CCC TAA 570 Ala Arg Val Phe AlaHis Gly Ala Ala Thr Leu Ser Pro                 160                 165MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVAQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP codingsequence: ATGCTGGGGA GCAGAGCTGT AATGCTGCTG TTGCTGCTGC CCTGGACAGCTCAGGGCAGA GCTGTGCCTG GGGGCAGCAG CCCTGCCTGG ACTCAGTGCC AGCAGCTTTCACAGAAGCTC TGCACACTGG CCTGGAGTGC ACATCCACTA GTGGGACACA TGGATCTAAGAGAAGAGGGA GATGAAGAGA CTACAAATGA TGTTCCCCAT ATCCAGTGTG GAGATGGCTGTGACCCCCAA GGACTCAGGG ACAACAGTCA GTTCTGCTTG CAAAGGATCC ACCAGGGTCTGATTTTTTAT GAGAAGCTGC TAGGATCGGA TATTTTCACA GGGGAGCCTT CTCTGCTCCCTGATAGCGCT GTGGCGCAGC TTCATGCCTC CCTACTGGGC CTCAGCCAAC TCCTGCAGCCTGAGGGTCAC CACTGGGAGA CTCAGCAGAT TCCAAGCCTC AGTCCCAGCC AGCCATGGCAGCGTCTCCTT CTCCGCTTCA AAATCCTTCG CAGCCTCCAG GCCTTTGTGG CTGTAGCCGCCCGGGTCTTT GCCCATGGAG CAGCAACCCT GAGTCCCTAA Rodent, e.g., mouse, IL-B30(SEQ ID NO: 3 and 4): CGCTTAGAAG TCGGACTACA GAGTTAGACT CAGAACCAAAGGAGGTGGAT AGGGGGTCCA 60 CAGGCCTGGT GCAGATCACA GAGCCAGCCA GATCTGAGAAGCAGGGAACA AG ATG 115                                                          Met                                                          −21 CTG GATTGC AGA GCA GTA ATA ATG CTA TGG CTG TTG CCC TGG GTC ACT 163 Leu Asp CysArg Ala Val Ile Met Leu Trp Leu Leu Pro Trp Val Thr−20                 −15                 −10                  −5 CAG GGCCTG GCT GTG CCT AGG AGT AGC AGT CCT GAC TGG GCT CAG TGC 211 Gln Gly LeuAla Val Pro Arg Ser Ser Ser Pro Asp Trp Ala Gln Cys                  1               5                  10 CAG CAG CTC TCTCGG AAT CTC TGC ATG CTA GCC TGG AAC GCA CAT GCA 259 Gln Gln Leu Ser ArgAsn Leu Cys Met Leu Ala Trp Asn Ala His Ala         15                  20                  25 CCA GCG GGA CAT ATGAAT CTA CTA AGA GAA GAA GAG GAT GAA GAG ACT 307 Pro Ala Gly His Met AsnLeu Leu Arg Glu Glu Glu Asp Glu Glu Thr     30                  35                  40 AAA AAT AAT GTG CCC CGTATC CAG TGT GAA GAT GGT TGT GAC CCA CAA 355 Lys Asn Asn Val Pro Arg IleGln Cys Glu Asp Gly Cys Asp Pro Gln 45                  50                  55                  60 GGA CTCAAG GAC AAC AGC CAG TTC TGC TTG CAA AGG ATC CGC CAA GGT 403 Gly Leu LysAsp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln Gly                 65                  70                  75 CTG GCT TTTTAT AAG CAC CTG CTT GAC TCT GAC ATC TTC AAA GGG GAG 451 Leu Ala Phe TyrLys His Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu             80                  85                  90 CCT GCT GTA CTCCCT GAT AGG CCC ATG GAG GAA CTT CAC ACC TCC CTA 499 Pro Ala Leu Leu ProAsp Ser Pro Met Glu Gln Leu His Thr Ser Leu         95                 100                 105 CTA GGA CTG AGC CAACTC CTC CAG GGA GAG GAT CAC CCC CGG GAG AGC 547 Leu Gly Leu Ser Gln LeuLeu Gln Pro Glu Asp His Pro Arg Glu Thr    110                 115                 120 CAA GAG ATG CCC AGC CTGAGT TCT AGT GAG GAG TGG GAG GGG CCC GTT 595 Gln Gln Met Pro Ser Leu SerSer Ser Gln Gln Trp Gln Arg Pro Leu125                 130                 135                 140 CTG CGTTGG AAG ATC CTT CGA AGC CTC CAG GCC TTT TTG GCC ATA GCT 643 Leu Arg SerLys Ile Leu Arg Ser Leu Gln Ala Phe Leu Ala Ile Ala                145                 150                 155 GCC CGG GTCTTT GCC CAC GGA GCA GCA ACT CTG ACT GAG CCC TTA GTG 691 Ala Arg Val PheAla His Gly Ala Ala Thr Leu Thr Glu Pro Leu Val            160                 165                 170 CCA ACA CGTTAAGGATGCC CAGGTTCCCA TGGCTACCAT GATAAGACTA 740 Pro Thr Ala         175ATCTATCACG CCAGACATCT ACCAGTTAAT TAACCCATTA GGACTTGTGC TGTTCTTGTT 800TCGTTTGTTT TGCGTGAAGG GCAAGGACAC CATTATTAAA GAGAAAAGAA ACAAACCCCA 860GAGCAGGCAC CTGGCTAGAG AAAGGAGCTG GAGAAGAAGA ATAAAGTCTC GAGCCCTTGG 920CCTTGGAAGC GGGCAAGCAG CTGCGTGGCC TGAGGGGAAG GGGGCGGTGG CATCGAGAAA 980CTGTGAGAAA ACCCAGACGA TGAGAAAAAG TGAGCCCAGG CTTTGGCCAT TATCTGTAAG 1040AAAAACAAGA AAAGGGGAAG ATTATACTTT CCTGGGTGGC TCAGGGAAAT GTGCAGATGC 1100ACAGTACTCC AGACAGCAGC TCTGTACCTG CCTGCTCTGT CCCTCAGTTC TAACAGAATC 1160TAGTCACTAA GAACTAACAG GACTACCAAT ACGAACTGAC AAA 1203MLDCRAVIMLWLLPWVTQGLAVPRSSSPDWAQCQQLSRNLCMLAWNAHAPAGHMNLLREEEDEETKNNVPRIQCEDGCDPQGLKDNSQFCLQRIRQGLAFYKHLLDSDIFKGEPALLPDSPMEQLHTSLLGLSQLLQPEDHPRETQQMPSLSSSQQWQRPLLRSKILRSLQAFLAIAARVFAHGAATLTEPLVPTA

The structural homology of IL-B30 to related cytokine proteins suggestsrelated function of this molecule. However, recognition of theassociation of the IL-12 p40 polypeptide with the IL-B30 polypeptideallows for biological assay of active p40/IL-B30 dimers. IL-12p40/IL-B30 compositions may be made up of either distinct polypeptidesrepresenting each of the individual polypeptides, or fusion constructsof IL-12 p40 with IL-B30. Observations indicate that the dimer iscapable of inducing interferon-γ (IFNγ) production by various celltypes, e.g., PBMC, suggesting biological functions for which the dimerwill be used. Moreover, experiments indicate that the IL-12 receptor β1subunit is a component of the receptor for the p40/IL-B30 dimer.

IFNγ activates macrophages, stimulating tumoricidal and microbicidalactivities. It also modulates class I and II MHC molecule expression,including up-regulation of class II molecules on monocytes/macrophagesand dendritic cells, and induces expression on epithelial, endothelial,and other cells, rendering them capable of antigen presentation. Thecytokine is a Th1-like cytokine which promotes the development ofTh1-like CD4+ T cells, but inhibits that of Th2-like T cells. It is apowerful and relatively specific inhibitor of IL-4-induced IgE and IgG4synthesis by B lymphocytes, although at higher concentrations itnon-specifically inhibits the production of all antibody isotypes. IFNγaugments cytotoxic immune responses against intracellular organisms andtumors mediated by NK cells and CTLs. Like IL-12, IFNγ has thepropensity to promote cell-mediated cytotoxic response while inhibitingallergic inflammation and IgE synthesis. See, e.g., Karupiah, (ed. 1997)Gamma Interferon in Antiviral Defense Chapman & Hall; Jaffe, (ed. 1992)Anti-Infective Applications of Interferon-Gamma Marcel Dekker (ISBN:0824786882); Sutterwala et al., (1999) J. Leukoc. Biol. 65:543–551;Billiau et al., (1998) Ann. NY Acad. Sci. 856:22–32; and Gessani et al.,(1998) Cytokine Growth Factor Rev. 9:117–123.

IL-B30 agonists, or antagonists, may also act as functional or receptorantagonists, e.g., which block IL-6 or IL-12 binding to their respectivereceptors, or mediating the opposite actions. Thus, IL-B30, or itsantagonists, may be useful in the treatment of abnormal medicalconditions, including immune disorders, e.g., T cell immunedeficiencies, chronic inflammation, or tissue rejection, or incardiovascular or neurophysiological conditions. Agonists would belikely to be used in a therapeutic context of enhancing cell mediatedimmunity, e.g., in anti-tumor, adjuvant, and anti-viral situations, orto antagonize allergic responses. Antagonists would likely be used inthe context of blocking such enhanced immunity, e.g., in cellularcontributions to autoimmune diseases or chronic inflammatory conditions.

The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The preferred embodiments would be from human, but other primate,or other species counterparts exist in nature. Additional sequences forproteins in other mammalian species, e.g., primates, canines, felines,and rodents, should also be available.

In particular, the association of the IL-12 p40 subunit with IL-B30 hasbeen confirmed. The IL-12 p40 and IL-B30 molecules should have evolvedtogether. If the two functionally associate, they might act together inthe fashion of IL-12. See, e.g., Trinchieri (1998) Adv. Immunol.70:83–243; Gately et al., (1998) Ann. Rev. Immunol. 16:495–521; andTrinchieri (1998) Int. Rev. Immunol. 16:365–396.

As a complex, however, the complex would be expected to interact withtwo tall signaling receptors in the cytokine receptor family. This hasbeen confirmed in the case of IL-12 receptor subunit β1. Other relatedreceptors can be tested for binding to the soluble complex. A series ofcells, e.g., BAF/3, that stably express various of these tall receptorscapable of signal transduction have been constructed.

The supernatants of transfectants of both IL-12 p40 and IL-B30 (or asingle combination construct) in the same cell, were used to test thesevarious cells to see if there is a proliferative or other signalingresponse. As such, most of the physiological effects of the cytokine maybe due to the complex of the proteins. As such, many of the descriptionsbelow of biology resulting from the cytokine may actually bephysiologically effected by the complex comprising the combination ofthe subunits.

The descriptions below may also be applied to the IL-12 p40/IL-B30complex. A fusion of the IL-12 p40 subunit with the IL-B30 wasconstructed, as, e.g., the hyper IL-6. See, e.g., Fischer et al., (1997)Nature Biotechnol. 15:142–145; Rakemann et al., (1999) J. Biol. Chem.274:1257–1266; and Peters et al., (1998) J. Immunol. 161:3575–3581;which are incorporated herein by reference. Moreover, matching of thecytokine complex with a receptor comprising the IL-12 receptor subunitβ1 allows for identification of antibodies to that subunit as a receptorantagonist of the cytokine complex.

II. Purified p40/IL-B30 Complex

Human IL-B30 amino acid sequence, is shown as one embodiment within SEQID NO: 2. Other naturally occurring nucleic acids which encode theprotein can be isolated by standard procedures using the providedsequence, e.g., PCR techniques, or by hybridization. These amino acidsequences, provided amino to carboxy, are important in providingsequence information for the cytokine subunit allowing fordistinguishing the protein antigen from other proteins and exemplifyingnumerous variants. Moreover, the peptide sequences allow preparation ofpeptides to generate antibodies to recognize segments, and nucleotidesequences allow preparation of oligonucleotide probes, both of which arestrategies for detection or isolation, e.g., cloning, of genes encodingsuch sequences.

As used herein, the term “human soluble IL-B30” shall encompass, whenused in a protein context, a protein having amino acid sequencecorresponding to a soluble polypeptide from SEQ ID NO: 2. Significantfragments thereof will often retain similar functions, e.g.,antigenicity. Preferred embodiments comprise a plurality of distinct,e.g., nonoverlapping, segments of the specified length. Typically, theplurality will be at least two, more usually at least three, andpreferably 5, 7, or even more. While the length minima may be recited,longer lengths, of various sizes, may be appropriate, e.g., one oflength 7, and two of length 12. Similar features apply to the IL-12 p40polypeptide, and to polynucleotides of either or both.

Binding components, e.g., antibodies, typically bind to an IL-12p40/IL-B30 complex with high affinity, e.g., at least about 100 nM,usually better than about 30 nM, preferably better than about 10 nM, andmore preferably at better than about 3 nM. Counterpart protein complexeswill be found in mammalian species other than human, e.g., otherprimates, ungulates, or rodents. Non-mammalian species should alsopossess structurally or functionally related genes and proteins, e.g.,birds or amphibians.

The term “polypeptide” as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids, e.g., 35, 40, 45, 50, etc. Such fragments may have endswhich begin and/or end at virtually all positions, e.g., beginning atresidues 1, 2, 3, etc., and ending at, e.g., 175, 174, 173, etc., in allpractical combinations for either the IL-B30 or the IL-12 p40 subunit.Particularly interesting peptides have ends corresponding to structuraldomain boundaries, e.g., helices A, B, C, and/or D of the IL-B30 or theIg domains of the IL-12 p40. See below.

The term “binding composition” refers to molecules that bind withspecificity to the IL-12 p40/IL-B30 complex, e.g., in anantibody-antigen interaction, but not to the individual componentsalone. The specificity may be more or less inclusive, e.g., specific toa particular embodiment, or to groups of related embodiments, e.g.,primate, rodent, etc. Depletion or absorptions can provide desiredselectivities, e.g., to deplete antibodies which bind to eitherpolypeptide component alone. Also provided are compounds, e.g.,proteins, which specifically associate with the IL-12 p40/IL-B30complex, including in a natural physiologically relevant protein-proteininteraction, either covalent or non-covalent. The molecule may be apolymer, or chemical reagent. A functional analog may be a protein withstructural modifications, or it may be a molecule which has a molecularshape which interacts with the appropriate binding determinants. Thecompounds may serve as agonists or antagonists of a receptor bindinginteraction, see, e.g., Goodman et al., (eds.), Goodman & Gilman's: ThePharmacological Bases of Therapeutics (current ed.) Pergamon Press.

Substantially pure, e.g., in a protein context, typically means that theprotein is free from other contaminating proteins, nucleic acids, orother biologicals derived from the original source organism. Purity maybe assayed by standard methods, typically by weight, and will ordinarilybe at least about 40% pure, generally at least about 50% pure, often atleast about 60% pure, typically at least about 80% pure, preferably atleast about 90% pure, and in most preferred embodiments, at least about95% pure. Carriers or excipients will often be added. A compositioncomprising a substantially pure IL-12 p40 and IL-B30 will not have largeamounts of extraneous polypeptides which are not naturally associatedwith the complex of the two polypeptides.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptides should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents. In particular, a complex made up of the association of thetwo polypeptides is preferred, as is a fusion composition.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein. Inother instances, a harsh detergent may be used to effect significantdenaturation.

An IL-B30 polypeptide that specifically binds to or that is specificallyimmunoreactive with an antibody, e.g., such as a polyclonal antibody,generated against a defined immunogen, e.g., such as an immunogenconsisting of an amino acid sequence of SEQ ID NO: 2 or fragmentsthereof or a polypeptide generated from the nucleic acid of SEQ ID NO: 1is typically determined in an immunoassay. Included within the metes andbounds of the present invention are those nucleic acid sequencesdescribed herein, including functional variants, that encodepolypeptides that selectively bind to polyclonal antibodies generatedagainst the prototypical IL-B30 polypeptide as structurally andfunctionally defined herein. The immunoassay typically uses a polyclonalantiserum which was raised, e.g., to a complex comprising a protein ofSEQ ID NO: 2. This antiserum is selected, or depleted, to have lowcrossreactivity against appropriate other closely related familymembers, preferably from the same species, and any such crossreactivityis removed by immunoabsorption or depletion prior to use in theimmunoassay. In particular, antibodies which bind to the IL-12 p40 orthe IL-B30 polypeptides alone are targets for immunodepletion.Appropriate selective serum preparations can be isolated, andcharacterized.

In order to produce antisera for use in an immunoassay, the complscomprising the protein, e.g., of SEQ ID NO: 2, is isolated as describedherein. For example, recombinant protein may be produced in a mammaliancell line. An appropriate host, e.g., an inbred strain of mice such asBalb/c, is immunized with the complex comprising a protein of SEQ ID NO:2 using a standard adjuvant, such as Freund's adjuvant, and a standardmouse immunization protocol (see Harlow and Lane). Alternatively, asubstantially full-length synthetic peptide construct derived from thesequences disclosed herein can be used as an immunogen. Polyclonal seraare collected and titered against the immunogen protein in animmunoassay, e.g., a solid phase immunoassay with the immunogenimmobilized on a solid support, along with appropriate depletions orselections. Polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against other closelyrelated family members, e.g., LIF, CT-1, CNTF, or other members of theIL-6 family, using a competitive binding immunoassay such as the onedescribed in Harlow and Lane, supra, at pages 570–573. Preferably atleast two individual IL-6/IL-12 family members are used in thisdetermination in conjunction with the target. These long chain cytokinefamily members can be produced as recombinant proteins and isolatedusing standard molecular biology and protein chemistry techniques asdescribed herein. Thus, antibody preparations can be identified orproduced having desired selectivity or specificity for subsets of IL-12p40/IL-B30 family members. Alternatively, antibodies may be preparedwhich bind to fusion polypeptide forms of the complex comprising theIL-12 p40 and IL-B30.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the fusion protein can beimmobilized to a solid support. Proteins added to the assay compete withthe binding of the selective antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theselective antisera to the immobilized protein is compared to the fusionprotein. The percent crossreactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% crossreactivity with each of the proteins listed above are selectedand pooled. The cross-reacting selective antibodies are then removedfrom the pooled antisera by immunoabsorption with the above-listedproteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen fusion protein. In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theselective antisera to the immobilized fusion protein is determined. Ifthe amount of the second protein required is less than twice the amountof the fusion protein that is required, then the second protein is saidto specifically bind to a selective antibody generated to the immunogen.

III. Physical Variants

This invention also encompasses complexes comprising proteins orpeptides having substantial amino acid sequence identity with the aminoacid sequences of the IL-12 p40/IL-B30 antigen. The variants includespecies, polymorphic, or allelic variants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham et al., (1970) J. Mol. Biol. 48:443–453;Sankoff et al., (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. The conservation may apply tobiological features, functional features, or structural features.Homologous amino acid sequences are typically intended to includenatural polymorphic or allelic and interspecies variations of a proteinsequence. Typical homologous proteins or peptides will have from 25–100%identity (if gaps can be introduced), to 50–100% identity (ifconservative substitutions are included) with the amino acid sequence ofthe IL-B30. Identity measures will be at least about 35%, generally atleast about 40%, often at least about 50%, typically at least about 60%,usually at least about 70%, preferably at least about 80%, and morepreferably at least about 90%.

The isolated IL-12 p40 or IL-B30 DNA can be readily modified bynucleotide substitutions, nucleotide deletions, nucleotide insertions,and inversions of short nucleotide stretches. These modifications resultin novel DNA sequences which encode these antigens, their derivatives,or proteins having similar physiological, immunogenic, antigenic, orother functional activity. These modified sequences can be used toproduce mutant antigens or to enhance expression. Enhanced expressionmay involve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant IL-B30” encompasses apolypeptide otherwise falling within the sequence identity definition ofthe IL-B30 as set forth above, but having an amino acid sequence whichdiffers from that of IL-B30 as normally found in nature, whether by wayof deletion, substitution, or insertion. This generally includesproteins having significant identity with a protein having sequence ofSEQ ID NO: 2, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the natural full-length disclosed sequences.Full-length sequences will typically be preferred, though truncatedversions will also be useful, likewise, genes or proteins found fromnatural sources are typically most desired. Similar concepts apply todifferent IL-B30 proteins, particularly those found in variouswarm-blooded animals, e.g., mammals and birds. These descriptions aregenerally meant to encompass various IL-B30 proteins, not limited to theparticular primate embodiments specifically discussed.

IL-12 p40 or IL-B30 mutagenesis can also be conducted by making aminoacid insertions or deletions. Substitutions, deletions, insertions, orany combinations may be generated to arrive at a final construct.Insertions include amino- or carboxy-terminal fusions. Randommutagenesis can be conducted at a target codon and the expressed mutantscan then be screened for the desired activity. Methods for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known in the art, e.g., by M13 primer mutagenesis orpolymerase chain reaction (PCR) techniques. See, e.g., Sambrook et al.,(1989); Ausubel et al., (1987 and Supplements); and Kunkel et al.,(1987) Methods in Enzymol. 154:367–382. Preferred embodiments include,e.g., 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, etc., preferablyconservative substitutions at the nucleotide or amino acid levels.Preferably the substitutions will be away from the conserved cysteines,and often will be in the regions away from the helical structuraldomains. Such variants may be useful to produce specific antibodies, andoften will share many or all biological properties. Recognition of thecytokine structure provides important insight into the structure andpositions of residues which may be modified to effect desired changes inreceptor interaction. Also, the interaction of the IL-12 p40 with theIL-B30 protein requires complementary structural features in theinteracting surface. Structural analysis will further allow predictionof the surface residues critical in both complex formation and complexto receptor interaction.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham et al., (1989) Science243:1330–1336; and O'Dowd et al., (1988) J. Biol. Chem. 263:15985–15992.

The phosphoramidite method described by Beaucage and Carruthers, (1981)Tetra. Letts. 22:1859–1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

Structural analysis can be applied to this gene, in comparison to theIL-6 family of cytokines. The family includes, e.g., IL-6, IL-11, IL-12,G-CSF, LIF, OSM, CNTF, and Ob. Alignment of the human and mouse IL-B30sequences with other members of the IL-6 family should allow definitionof structural features. In particular, β-sheet and α-helix residues canbe determined using, e.g., RASMOL program, see Bazan et al., (1996)Nature 379:591; Lodi et al., (1994) Science 263:1762–1766; Sayle andMilner-White, (1995) TIBS 20:374–376; and Gronenberg et al., (1991)Protein Engineering 4:263–269. See, also, Wilkins et al., (eds. 1997)Proteome Research: New Frontiers in Functional Genomics Springer-Verlag,N.Y. Preferred residues for substitutions include the surface exposedresidues which would be predicted to interact with receptor. Otherresidues which should conserve function will be conservativesubstitutions, particularly at a position far from the surface exposedresidues.

IV. Functional Variants

The blocking of physiological response to the IL-12 p40/IL-B30 complexesmay result from the competitive inhibition of binding of the ligand toits receptor. Identification of one subunit of the receptor allows forfurther characterization, as described, and use of antibodies to thatsubunit to block binding and/or signaling with the complex.

In vitro assays of the present invention will often use isolatedcomplex, protein, soluble fragments comprising receptor binding segmentsof these proteins, or fragments attached to solid phase substrates.These assays will also allow for the diagnostic determination of theeffects of either binding segment mutations and modifications, orcytokine mutations and modifications, e.g., IL-12 p40/IL-B30 complexanalogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the cytokine complex, orreceptor binding fragments compete with a test compound.

“Derivatives” of IL-12 p40/IL-B30 antigens include amino acid sequencemutants from naturally occurring forms, glycosylation variants, andcovalent or aggregate conjugates with other chemical moieties. Covalentderivatives can be prepared by linkage of functionalities to groupswhich are found in IL-12 p40/IL-B30 complex amino acid side chains or atthe N- or C-termini, e.g., by standard means. See, e.g., Lundblad andNoyes, (1988) Chemical Reagents for Protein Modification, vols. 1–2, CRCPress, Inc., Boca Raton, Fla.; Hugli, (ed. 1989) Techniques in ProteinChemistry, Academic Press, San Diego, Calif.; and Wong, (1991) Chemistryof Protein Conjugation and Cross Linking, CRC Press, Boca Raton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein, (1987) Ann. Rev. Biochem. 56:497–534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between the IL-12 p40 and IL-B30 are also provided.Many cytokine receptors or other surface proteins are multimeric, e.g.,homodimeric entities, and a repeat construct may have variousadvantages, including lessened susceptibility to proteolytic cleavage.Typical examples are fusions of a reporter polypeptide, e.g.,luciferase, with a segment or domain of a protein, e.g., areceptor-binding segment, so that the presence or location of the fusedligand may be easily determined. See, e.g., Dull et al., U.S. Pat. No.4,859,609. Other gene fusion partners include bacterial β-galactosidase,trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase,yeast alpha mating factor, and detection or purification tags such as aFLAG sequence of His6 sequence. See, e.g., Godowski et al., (1988)Science 241:812–816. Fusion constructs with other therapeutic entities,e.g., which are to be coadministered, but proteolytically cleaved, arealso provided.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1–3, Cold Spring Harbor Laboratory; and Ausubel et al., (eds.1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield, (1963) J. Amer. Chem. Soc. 85:2149–2156; Merrifield, (1986)Science 232:341–347; Atherton et al., (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant, (1992)Synthetic Peptides: A User's Guide, W. H. Freeman, NY. Refolding methodsmay be applicable to synthetic proteins.

This invention also contemplates the use of derivatives of IL-12 p40 orIL-B30 proteins other than variations in amino acid sequence orglycosylation. Such derivatives may involve covalent or aggregativeassociation with chemical moieties or protein carriers. Covalent oraggregative derivatives will be useful as immunogens, as reagents inimmunoassays, or in purification methods such as for affinitypurification of binding partners, e.g., other antigens. An IL-12 p40 orIL-B30 can be immobilized by covalent bonding to a solid support such ascyanogen bromide-activated SEPHAROSE, by methods which are well known inthe art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-IL-12 p40 or IL-B30 antibodies or an alternative bindingcomposition. The IL-12 p40, IL-B30, or fusion proteins can also belabeled with a detectable group, e.g., for use in diagnostic assays.Purification of IL-12 p40/IL-B30 complex may be effected by animmobilized antibody to either polypeptide or sequence component orcomplementary binding partner, e.g., binding portion of a receptor.

A solubilized IL-12 p40/IL-B30 polypeptide or fragment of this inventioncan be used as an immunogen for the production of antisera or antibodiesspecific for binding. Purified antigen can be used to screen monoclonalantibodies or antigen-binding fragments, encompassing antigen bindingfragments of natural antibodies, e.g., Fab, Fab′, F(ab)₂, etc. PurifiedIL-12 p40/IL-B30 antigens can also be used as a reagent to detectantibodies generated in response to the presence of elevated levels ofthe cytokine complex, which may be diagnostic of an abnormal or specificphysiological or disease condition. This invention contemplatesantibodies raised against amino acid sequences encoded by nucleotidesequence shown in SEQ ID NO: 1, or fragments of proteins containing it.In particular, this invention contemplates antibodies having bindingaffinity to or being raised against specific domains, e.g., helices A,B, C, or D of the IL-B30, or the Ig domains of the IL-12 p40.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis willestablish that similar genetic entities exist in other mammals. It islikely that IL-B30s are widespread in species variants, e.g., rodents,lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species or polymorphic variantsof them. In particular, the present invention provides useful probes foridentifying additional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof an IL-B30, e.g., either species types or cells which lackcorresponding proteins and exhibit negative background activity. Thisshould allow analysis of the function of IL-B30 in comparison tountransformed control cells.

Dissection of critical structural elements which effect the variousphysiological functions mediated through these antigens is possibleusing standard techniques of modern molecular biology, particularly incomparing members of the related class. See, e.g., the homolog-scanningmutagenesis technique described in Cunningham et al., (1989) Science243:1339–1336; and approaches used in O'Dowd et al., (1988) J. Biol.Chem. 263:15985–15992; and Lechleiter et al., (1990) EMBO J.9:4381–4390.

Intracellular functions would probably involve receptor signaling.However, protein internalization may occur under certain circumstances,and interaction between intracellular components and cytokine may occur.Specific segments of interaction of IL-B30 with interacting componentsmay be identified by mutagenesis or direct biochemical means, e.g.,cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of IL-B30 will be pursued.The controlling elements associated with the antigens should exhibitdifferential physiological, developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest.

Structural studies of the IL-B30 antigens will lead to design of newantigens, particularly analogs exhibiting agonist or antagonistproperties on the molecule. This can be combined with previouslydescribed screening methods to isolate antigens exhibiting desiredspectra of activities.

V. Antibodies

Antibodies can be raised to various epitopes of the p40/IL-B30 proteins,including species, polymorphic, or allelic variants, and fragmentsthereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to IL-B30s ineither their active forms or in their inactive forms, including nativeor denatured versions. Anti-idiotypic antibodies are also contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective IL-B30s, or screened for agonistic orantagonistic activity, e.g., mediated through a receptor. Antibodies maybe agonistic or antagonistic, e.g., by sterically blocking binding to areceptor. These monoclonal antibodies will usually bind with at least aK_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 100 μM, more typically at least about 30 μM, preferably atleast about 10 μM, and more preferably at least about 3 μM or better.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingto a receptor. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying IL-B30 protein or its receptors. See, e.g., Chan, (ed. 1987)Immunology: A Practical Guide, Academic Press, Orlando, Fla.; Price andNewman, (eds. 1991) Principles and Practice of Immunoassay, StocktonPress, N.Y.; and Ngo, (ed. 1988) Nonisotopic Immunoassay, Plenum Press,N.Y. Cross absorptions or other tests will identify antibodies whichexhibit various spectra of specificities, e.g., unique or shared speciesspecificities.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding, e.g., to a receptor which may elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides so that when the antibody binds toantigen, a cell expressing it, e.g., on its surface, is killed. Further,these antibodies can be conjugated to drugs or other therapeutic agents,either directly or indirectly by means of a linker, and may effect drugtargeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner, (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams et al.,(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane, (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites et al., (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane, (1988) Antibodies: A Laboratory Manual,CSH Press; Goding, (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein, (1975) in Nature 256:495–497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse et al., (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275–1281; and Ward et al.,(1989) Nature 341:544–546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore et al.,U.S. Pat. No. 4,642,334; and Queen et al., (1989) Proc. Natl. Acad. Sci.USA 86:10029–10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.,(1984) Meth. Enzymol. 104:3–55. Conversely, protein can be used fordepletion or cross absorptions to prepare selectively specific bindingcompositions.

Antibodies raised against each IL-B30 will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding both IL-12 p40and IL-B30, e.g., from a natural source. Typically, it will be useful inisolating genes from a mammal, and similar procedures will be applied toisolate genes from other species, e.g., warm-blooded animals, such asbirds and mammals. Cross hybridization will allow isolation of IL-12 p40or IL-B30 from the same, e.g., polymorphic variants, or other species. Anumber of different approaches will be available to successfully isolatea suitable nucleic acid clone. Such genes allow construction ofcoexpression constructs or fusion constructs.

The purified protein or polypeptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan, (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane, (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

For example, a specific binding composition could be used for screeningof an expression library made from a cell line which expresses bothIL-12 p40 and IL-B30. Screening of intracellular expression can beperformed by various staining or immunofluorescence procedures. Bindingcompositions could be used to affinity purify or sort out cellsexpressing a surface fusion protein.

The peptide segments can also be used to select or identify appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., GenBank and SEQ ID NO: 1. In combination with polymerase chainreaction (PCR) techniques, synthetic oligonucleotides will be useful inselecting correct clones from a library. Complementary sequences willalso be used as probes, primers, or antisense strands. Various fragmentsshould be particularly useful, e.g., coupled with anchored vector orpoly-A complementary PCR techniques or with complementary DNA of otherpeptides.

This invention contemplates use of isolated DNA or fragments to encode abiologically active complex of the corresponding IL-12 p40 and IL-B30polypeptide, particularly lacking the portion coding the untranslatedportions of the described sequences. In addition, this invention coversisolated or recombinant DNA which encodes a biologically active fusionprotein or polypeptide and which is capable of hybridizing underappropriate conditions with the DNA sequences described herein. Saidbiologically active protein or polypeptide can be an intact antigen, orfragment, and have an amino acid sequence disclosed in, e.g., SEQ ID NO:2, particularly a mature, secreted polypeptide. Further, this inventioncovers the use of isolated or recombinant DNA, or fragments thereof,which encode proteins which exhibit high identity to a secreted IL-12p49/IL-B30 complex. The isolated DNA can have the respective regulatorysequences in the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-Aaddition signals, and others. Alternatively, expression may be effectedby operably linking a coding segment to a heterologous promoter, e.g.,by inserting a promoter upstream from an endogenous gene. See, e.g.,Treco et al., WO96/29411 or U.S. Ser. No. 08/406,030.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other extraneouscomponents which naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule,e.g., distinct from an isolated chromosome. Generally, the nucleic acidwill be in a vector or fragment less than about 50 kb, usually less thanabout 30 kb, typically less than about 10 kb, and preferably less thanabout 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesor polymorphic variants.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, etc., includinghundreds and/or thousands.

A DNA which codes for an IL-B30 protein will be particularly useful toidentify genes, mRNA, and cDNA species which code for related or similarproteins, as well as DNAs which code for homologous proteins fromdifferent species. There will be homologs in other species, includingprimates, rodents, canines, felines, and birds. Various IL-B30 proteinsshould be homologous and are encompassed herein. However, even proteinsthat have a more distant evolutionary relationship to the antigen canreadily be isolated under appropriate conditions using these sequencesif they are sufficiently homologous. Primate IL-B30 proteins are ofparticular interest. Likewise with the IL-12 p40, which proteins areprime targets for the fusion constructs or combination compositions.

Recombinant clones derived from the genomic sequences, e.g., containingintrons, will be useful for transgenic studies, including, e.g.,transgenic cells and organisms, and for gene therapy. See, e.g.,Goodnow, (1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia ofImmunology, Academic Press, San Diego, pp. 1502–1504; Travis, (1992)Science 256:1392–1394; Kuhn et al., (1991) Science 254:707–710; Capecchi(1989) Science 244:1288; Robertson, (ed. 1987) Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, IRL Press, Oxford; andRosenberg, (1992) J. Clinical Oncology 10:180–199.

Substantial homology, e.g., identity, in the nucleic acid sequencecomparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least about 58%,ordinarily at least about 65%, often at least about 71%, typically atleast about 77%, usually at least about 85%, preferably at least about95 to 98% or more, and in particular embodiments, as high as about 99%or more of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, typically using a sequenceof IL-12 p40 and/or IL-B30, e.g., in SEQ ID NO: 1. Typically, selectivehybridization will occur when there is at least about 55% identity overa stretch of at least about 30 nucleotides, preferably at least about75% over a stretch of about 25 nucleotides, and most preferably at leastabout 90% over about 20 nucleotides. See, Kanehisa, (1984) Nuc. AcidsRes. 12:203–213. The length of identity comparison, as described, may beover longer stretches, and in certain embodiments will be over a stretchof at least about 17 nucleotides, usually at least about 28 nucleotides,typically at least about 40 nucleotides, and preferably at least about75 to 100 or more nucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., more typically inexcess of about 60 or 65° C., and preferably in excess of about 70° C.Stringent salt conditions will ordinarily be less than about 1000 mM,usually less than about 400 mM, typically less than about 250 mM,preferably less than about 150 mM, including about 100, 50, or even 20mM. However, the combination of parameters is much more important thanthe measure of any single parameter. See, e.g., Wetmur and Davidson,(1968) J. Mol. Biol. 31:349–370. Hybridization under stringentconditions should give a background of at least 2-fold over background,preferably at least 3–5 or more.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman, (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman andWunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity methodof Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle, (1987) J. Mol. Evol.35:351–360. The method used is similar to the method described byHiggins and Sharp, (1989) CABIOS 5:151–153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul et al., (1990) J. Mol. Biol. 215:403–410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, (1993) Proc. Natl. Acad.Sci. USA 90:5873–5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, e.g., where the two peptides differ only by conservativesubstitutions. Another indication that two nucleic acid sequences aresubstantially identical is that they hybridize to each other understringent conditions, as described below.

IL-B30 from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

VII. Making p40/IL-B30 Combinations; Mimetics

DNA which encodes the IL-12 p40 or IL-B30 or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or screeninggenomic libraries prepared from a wide variety of cell lines or tissuesamples. See, e.g., Okayama and Berg, (1982) Mol. Cell. Biol. 2:161–170;Gubler and Hoffman, (1983) Gene 25:263–269; and Glover, (ed. 1984) DNACloning: A Practical Approach, IRL Press, Oxford. Alternatively, thesequences provided herein provide useful PCR primers or allow syntheticor other preparation of suitable genes encoding an IL-12 p40 or IL-B30;including naturally occurring embodiments.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length IL-12 p40 and IL-B30 or fragments which can,in turn, e.g., be used to generate polyclonal or monoclonal antibodies;for binding studies; for construction and expression of modifiedmolecules; and for structure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels et al., (1985 and Supplements) Cloning Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez et al., (eds. 1988) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See, e.g., Rodriguez et al., Chapter 10, pp. 205–236; Balbasand Bolivar, (1990) Methods in Enzymology 185:14–37; and Ausubel et al.,(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Coexpression of the two coding sequences is particularly of interestherein.

Representative examples of suitable expression vectors include pCDNA1;pCD, see Okayama et al., (1985) Mol. Cell Biol. 5:1136–1142; pMClneoPoly-A, see Thomas et al., (1987) Cell 51:503–512; and a baculovirusvector such as pAC 373 or pAC 610. See, e.g., Miller, (1988) Ann. Rev.Microbiol. 42:177–199.

It will often be desired to express an IL-12 p40 and/or IL-B30polypeptide in a system which provides a specific or definedglycosylation pattern. See, e.g., Luckow and Summers, (1988)Bio/Technology 6:47–55; and Kaufman, (1990) Meth. Enzymol. 185:487–511.

The IL-12 p40 and/or IL-B30, or a fragment thereof, may be engineered tobe phosphatidyl inositol (PI) linked to a cell membrane, but can beremoved from membranes by treatment with a phosphatidyl inositolcleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. Thisreleases the antigen in a biologically active form, and allowspurification by standard procedures of protein chemistry. See, e.g.,Low, (1989) Biochim. Biophys. Acta 988:427–454; Tse et al., (1985)Science 230:1003–1008; and Brunner et al., (1991) J. Cell Biol.114:1275–1283.

Now that the IL-12 p40 and IL-B30 have been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young, (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky, (1984) The Practice ofPeptide Synthesis, Springer-Verlag, New York; Bodanszky, (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York; andVillafranca, (ed. 1991) Techniques in Protein Chemistry II, AcademicPress, San Diego, Calif.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in IL-12p40/IL-B30 complex mediated conditions, or below in the description ofkits for diagnosis. The gene may be useful in forensic sciences, e.g.,to distinguish rodent from human, or as a marker to distinguish betweendifferent cells exhibiting differential expression or modificationpatterns. The provided compositions are useful reagents for, e.g., invitro assays, scientific research, and the synthesis or manufacture ofnucleic acids, polypeptides, or antibodies.

This invention also provides reagents with significant commercial and/ortherapeutic potential. The IL-12 p40/IL-B30 complex (naturally occurringor recombinant), fragments thereof, and antibodies thereto, along withcompounds identified as having binding affinity to the complex orindividual components thereof, should be useful as reagents for teachingtechniques of molecular biology, immunology, or physiology. Appropriatekits may be prepared with the reagents, e.g., in practical laboratoryexercises in production or use of proteins, antibodies, cloning methods,histology, etc.

The reagents will also be useful in the treatment of conditionsassociated with abnormal physiology or development, includinginflammatory conditions. They may be useful in vitro tests for presenceor absence of interacting components, which may correlate with successof particular treatment strategies. In particular, modulation ofphysiology of various, e.g., hematopoietic or lymphoid, cells will beachieved by appropriate methods for treatment using the compositionsprovided herein. See, e.g., Thomson, (1994; ed.) The Cytokine Handbook(2d ed.) Academic Press, San Diego; Metcalf and Nicola, (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman, (1991) Human Cytokines Blackwell Pub.

Observations that the cytokine complex can induce IFNγ levels providesuseful insight into therapeutic potential. In particular, IFNγproduction results in enhanced cell mediated immunity. See, e.g., Paul,(1998) Fundamental Immunology (4th ed.) Raven Press, NY.; and Delves andRoitt (eds. 1998) The Encyclopedia of Immunology Academic Press (ISBN:0122267656). Thus, enhancement of cellular responses will be useful incontexts to enhance anti-tumor activity, enhance vaccine responses (bothhumoral and cellular immunity), enhance anti-viral effects, and toantagonize allergic responses in certain windows of development. See,e.g, Rose and Mackay (eds. 1998) The Autoimmune Diseases (3d ed.)Acadmeic Press, San Diego; and Kay, (ed. 1997) Allergy and AllergicDiseases Blackwell Science, Malden Mass. Conversely, antagonists wouldbe used to block or prevent such IFNγ enhancement, thereby reducing thestrength or intensity of the cellular enhancement. Such may be usefulin, e.g., autoimmune situations (such as multiple sclerosis orpsoriasis) or chronic inflammatory conditions (such as rheumatoidarthritis or inflammatory bowel disease). See, e.g., Samter et al.,(eds.) Immunological Diseases vols. 1 and 2, Little, Brown and Co. Theinitial results suggest that the role of the p40/IL-B30 is more criticalin the maintenance of the chronic inflammatory condition. Thus, blockagemay be effective after initial development of the condition.

With such therapeutic targets, the agonists or antagonists will becombined with existing therapeutics, e.g., with other modulators ofinflammation. Thus, the agonists will often be combined, e.g., withIL-18, IL-12, radiation or chemotherapy treatments, vaccine adjuvants,and/or anti-viral therapeutics. Alternatively, the antagonists may becombined with TNFα antagonists, IL-12 antagonists, with IL-10, and/orsteroids. Viral homologs of the cytokines might also be used.

For example, a disease or disorder associated with abnormal expressionor abnormal signaling by an IL-12 p40/IL-B30 should be a likely targetfor an agonist or antagonist. The new cytokine should play a role inregulation or development of hematopoietic cells, e.g., lymphoid cells,which affect immunological responses, e.g., inflammation and/orautoimmune disorders. Alternatively, it may affect vascular physiologyor development, or neuronal effects. Timing of administration of thetherapeutic relative to initiation or maintenance of the condition mayalso be important. In particular, the cytokine complex should mediate,in various contexts, cytokine synthesis by the cells, proliferation,etc. Antagonists of IL-12 p40/IL-B30, such as mutein variants of anaturally occurring form or blocking antibodies, may provide a selectiveand powerful way to block immune responses, e.g., in situations asinflammatory or autoimmune responses. See also Samter et al., (eds.)Immunological Diseases vols. 1 and 2, Little, Brown and Co.

Particular targets for therapeutic application include, e.g., lungconditions, both asthma and fibrosis, in EAE models (which may be usefulmodels for multiple sclerosis), diabetes, and gut inflammations. See,e.g., Barnes et al., (1998) Mol. Med. Today 4:452–458; Pauwels et al.,(1998) Clin. Exp. Allergy Aug. 28 Suppl 3:1–5; Durham, (1998) Clin. Exp.Allergy Jun. 28 Suppl 2:11–16; Leung, (1997) Pediatr. Res. 42:559–568;Pretolani et al., (1997) Res. Immunol. 148:33–38; Lamkhioued et al.,(1996) Ann. NY Acad. Sci. 796:203–208; Erb et al., (1996) Immunol. Cell.Biol. 74:206–208; and Anderson et al., (1994) Trends Pharmacol. Sci.15:324–332 for asthma; Coker et al., (1998) Eur. Respir. J.11:1218–1221; and Bienkowski et al., (1995) Proc. Soc. Exp. Biol. Med.209:118–140 for lung fibrosis; Pearson and McDevitt, (1999) Curr. Top.Microbiol. Immunol. 238:79–122; Miller and Shevach, (1998) Res. Immunol.149:753–759; Hoffman and Karpus, (1998) Res. Immunol. 149:790–794 (withdiscussion 846–847 and 855–860); Segal, (1998) Res. Immunol. 149:811–820(with discussion 850–851 and 855–860); Liblau et al., (1997) Immunol.Today 18:599–604; Gold et al., (1997) Crit. Rev. Immunol. 17:507–510;Spack, (1997) Crit. Rev. Immunol. 17:529–536; and Leonard et al., (1997)Crit. Rev. Immunol. 17:545–553 for EAE models (for multiple sclerosis);Almawi et al., (1999) J. Clin. Endocrinol. Metab. 84:1497–1502;Rabinovitch et al., (1998) Biochem. Pharmacol. 55:1139–1149; andRabinovitch, (1998) Diabetes Metab. Rev. 14:129–151 for diabetes; andLeach et al., (1999) Toxicol. Pathol. 27:123–133; Braun et al., (1999)Curr. Opin. Rheumatol. 11:68–74; Rugtveit et al., (1997)Gastroenterology 112:1493–1505; Strober et al., (1997) Immunol. Today18:61–64; and Ford et al., (1996) Semin. Pediatr. Surg. 5:155–159 forgut/intestinal inflammatory conditions.

The p40/IL-B30 stimulation of memory activated cells results inphenotypic changes which include adhesion molecules. CD69L is highlyexpressed following stimulation with p40/IL-B30, and CD54 isdramatically decreased. These changes in expression of adhesionmolecules may allow modulating memory cells to enter the T/DC cell richregion of primary and secondary lymph nodes, e.g., via high endothelialvenules (HEV). The memory cells are also primed to become sensitive toIL-12 stimulation. Thus, rapid and high IFN production would quicklyfollow IL-12 induction by antigen. Thus p40/IL-B30 may accelerate animmune response by memory cells, either by increasing response rate,increasing memory cell numbers, or both. The p40/IL-B30 may havedifferential effects specific for memory cells, with lesser or no effecton naive cells. Conversely, in many chronic inflammatory conditions,e.g., rheumatoid arthritis, inflammatory bowel disease, psoriasis, etc.,the active lesions are dependent upon memory CD45Rb^(low) cells. Assuch, antagonists may effectively block the chronic phase of such aninflammatory condition.

Various abnormal conditions are known in each of the cell types shown toproduce both IL-12 p40 and/or IL-B30 mRNA by Northern blot analysis. SeeBerkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co.,Rahway, N.J.; Thorn et al., Harrison's Principles of Internal Medicine,McGraw-Hill, N.Y.; and Weatherall et al., (eds.) Oxford Textbook ofMedicine, Oxford University Press, Oxford. Many other medical conditionsand diseases involve activation by macrophages or monocytes, and many ofthese will be responsive to treatment by an agonist or antagonistprovided herein. See, e.g., Stites and Terr (eds. 1991) Basic andClinical Immunology Appleton and Lange, Norwalk, Connecticut; and Samteret al. (eds.), Immunological Diseases Little, Brown and Co. Theseproblems should be susceptible to prevention or treatment usingcompositions provided herein.

The IL-12 p40/IL-B30 cytokine complex, antagonists, antibodies, etc.,can be purified and then administered to a patient, veterinary or human.These reagents can be combined for therapeutic use with additionalactive or inert ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, e.g., immunogenic adjuvants, along withphysiologically innocuous stabilizers, excipients, or preservatives.These combinations can be sterile filtered and placed into dosage formsas by lyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies orbinding fragments thereof, including forms which are not complementbinding.

Drug screening using IL-12 p40/IL-B30, fusion protein, or fragmentsthereof, can be performed to identify compounds having binding affinityto or other relevant biological effects on IL-12 p40/IL-B30 functions,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if a candidate compound hasintrinsic stimulating activity and is therefore a blocker or antagonistin that it blocks the activity of the cytokine complex. Likewise, acompound having intrinsic stimulating activity can activate the signalpathway and is thus an agonist in that it simulates the activity of thecytokine complex. This invention further contemplates the therapeuticuse of blocking antibodies to IL-12 p40, IL-B30, or the complex, asantagonists and of stimulatory antibodies as agonists. This approachshould be particularly useful with other IL-12 p40 or IL-B30 speciesvariants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman et al., (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer, (1990)Science 249:1527–1533.

IL-12 p40, IL-B30, cytokine complex, fusion proteins, fragments thereof,and antibodies to it or its fragments, antagonists, and agonists, may beadministered directly to the host to be treated or, depending on thesize of the compounds, it may be desirable to conjugate them to carrierproteins such as ovalbumin or serum albumin prior to theiradministration. Therapeutic formulations may be administered in manyconventional dosage formulations. While it is possible for the activeingredient to be administered alone, it is preferable to present it as apharmaceutical formulation. Formulations typically comprise at least oneactive ingredient, as defined above, together with one or moreacceptable carriers thereof. Each carrier should be bothpharmaceutically and physiologically acceptable in the sense of beingcompatible with the other ingredients and not injurious to the patient.Formulations include those suitable for oral, rectal, nasal, topical, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by many methods wellknown in the art of pharmacy. See, e.g., Gilman et al., (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa.; Avis et al., (eds. 1993)Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New York;Lieberman et al., (eds. 1990) Pharmaceutical Dosage Forms: Tablets,Dekker, New York; and Lieberman et al., (eds. 1990) PharmaceuticalDosage Forms: Disperse Systems, Dekker, New York. The therapy of thisinvention may be combined with or used in association with other agents,e.g., other cytokines, including IL-6 or G-CSF, or their respectiveantagonists.

Both naturally occurring and recombinant forms of the IL-B30s of thisinvention are particularly useful in kits and assay methods which arecapable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor et al., (1991) Science 251:767–773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble IL-12 p40/IL-B30 cytokine complex asprovided by this invention.

Other methods can be used to determine the critical residues in IL-12p40/IL-B30 complex-receptor interactions. Mutational analysis can beperformed, e.g., see Somoza et al., (1993) J. Exptl. Med. 178:549–558,to determine specific residues critical in the interaction and/orsignaling. PHD (Rost and Sander, (1994) Proteins 19:55–72) and DSC (Kingand Sternberg, (1996) Protein Sci. 5:2298–2310) can provide secondarystructure predictions of α-helix (H), β-strand (E), or coil (L). HelicesA and D are typically most important in receptor interaction, with the Dhelix the more important region.

For example, antagonists can normally be found once the antigen and/orreceptor has been structurally defined, e.g., by tertiary structuredata. Testing of potential interacting analogs is now possible upon thedevelopment of highly automated assay methods using a purified IL-12p40/IL-B30 complex. In particular, new agonists and antagonists will bediscovered by using screening techniques described herein. Of particularimportance are compounds found to have a combined binding affinity for aspectrum of IL-12 p40/IL-B30 molecules, e.g., compounds which can serveas antagonists for species variants of the cytokine complex.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing an IL-20 p40/IL-B30. Cells may be isolated which express anIL-12 p40/IL-B30 in isolation from other molecules. Such cells, eitherin viable or fixed form, can be used for standard binding partnerbinding assays. See also, Parce et al., (1989) Science 246:243–247; andOwicki et al., (1990) Proc. Natl. Acad. Sci. USA 87:4007–4011, whichdescribe sensitive methods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto an IL-12 p40/IL-B30 and is described in detail in Geysen, EuropeanPatent Application 84/03564, published on Sep. 13, 1984. First, largenumbers of different small peptide test compounds are synthesized on asolid substrate, e.g., plastic pins or some other appropriate surface,see Fodor et al., (1991). Then all the pins are reacted withsolubilized, unpurified or solubilized, purified p40/IL-B30, and washed.The next step involves detecting bound p40/IL-B30.

Rational drug design may also be based upon structural studies of themolecular shapes of the p40/IL-B30 and other effectors or analogs.Effectors may be other proteins which mediate other functions inresponse to binding, or other proteins which normally interact withp40/IL-B30, e.g., a receptor. One means for determining which sitesinteract with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions, as modeled, e.g., against othercytokine-receptor models. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson, (1976)Protein Crystallography, Academic Press, New York.

IX. Kits

This invention also contemplates use of p40/IL-B30 proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of another p40/IL-B30 orbinding partner. Typically the kit will have a compartment containingeither a defined p40, p40/IL-B30, or IL-B30 peptide or gene segment or areagent which recognizes one or the other, e.g., p40/IL-B30 fusionfragments or antibodies.

A kit for determining the binding affinity of a test compound to anIL-12 p40/IL-B30 would typically comprise a test compound; a labeledcompound, for example a binding partner or antibody having known bindingaffinity for p40/IL-B30; a source of p40/IL-B30 (naturally occurring orrecombinant); and a means for separating bound from free labeledcompound, such as a solid phase for immobilizing the molecule. Oncecompounds are screened, those having suitable binding affinity to theantigen can be evaluated in suitable biological assays, as are wellknown in the art, to determine whether they act as agonists orantagonists to the p40/IL-B30 signaling pathway. The availability ofrecombinant IL-12 p40/IL-B30 fusion polypeptides also provide welldefined standards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., a p40/IL-B30in a sample would typically comprise a labeled compound, e.g., bindingpartner or antibody, having known binding affinity for the antigen, asource of cytokine (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, e.g., a solid phase forimmobilizing the p40/IL-B30. Compartments containing reagents, andinstructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for thep40/IL-B30 or fragments are useful in diagnostic applications to detectthe presence of elevated levels of p40, IL-B30, p40/IL-B30, and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescence, cell cultures, body fluids, and further caninvolve the detection of antigens related to the antigen in serum, orthe like. Diagnostic assays may be homogeneous (without a separationstep between free reagent and antigen-binding partner complex) orheterogeneous (with a separation step). Various commercial assays exist,such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassaytechnique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), andthe like. See, e.g., Van Vunakis et al., (1980) Meth Enzymol. 70:1–525;Harlow and Lane, (1980) Antibodies: A Laboratory Manual, CSH Press, NY;and Coligan et al., (eds. 1993) Current Protocols in Immunology, Greeneand Wiley, NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a p40/IL-B30, as such may be diagnostic of variousabnormal states. For example, overproduction of p40/IL-B30 may result inproduction of various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal activation or differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeledp40/IL-B30 is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In many of these assays, the binding partner, testcompound, p40/IL-B30, or antibodies thereto can be labeled eitherdirectly or indirectly. Possibilities for direct labeling include labelgroups: radiolabels such as ¹²⁵I, enzymes such as peroxidase andalkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475)capable of monitoring the change in fluorescence intensity, wavelengthshift, or fluorescence polarization. Possibilities for indirect labelinginclude biotinylation of one constituent followed by binding to avidincoupled to one of the above label groups.

There are also numerous methods of separating the bound from the freep40/IL-B30, or alternatively the bound from the free test compound. Thep40/IL-B30 can be immobilized on various matrixes followed by washing.Suitable matrixes include plastic such as an ELISA plate, filters, andbeads. See, e.g., Coligan et al., (eds. 1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle et al., (1984)Clin. Chem. 30:1457–1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of ap40/IL-B30. These sequences can be used as probes for detecting levelsof the p40 or IL-B30 messages in samples from patients suspected ofhaving an abnormal condition, e.g., inflammatory or autoimmune. Sincethe cytokine may be a marker or mediator for activation, it may beuseful to determine the numbers of activated cells to determine, e.g.,when additional therapy may be called for, e.g., in a preventativefashion before the effects become and progress to significance. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. See, e.g.,Langer-Safer et al., (1982) Proc. Natl. Acad. Sci. 79:4381–4385; Caskey,(1987) Science 236:962–967; and Wilchek et al., (1988) Anal. Biochem.171:1–32.

Diagnostic kits which also test for the qualitative or quantitativeexpression of other molecules are also contemplated. Diagnosis orprognosis may depend on the combination of multiple indications used asmarkers. Thus, kits may test for combinations of markers. See, e.g.,Viallet et al., (1989) Progress in Growth Factor Res. 1:89–97. Otherkits may be used to evaluate other cell subsets.

X. Isolating a p40/IL-B30 Receptor

Having isolated a ligand of a specific ligand-receptor interaction,methods exist for isolating the receptor. See, Gearing et al., (1989)EMBO J. 8:3667–3676. For example, means to label the IL-B30 cytokinewithout interfering with the binding to its receptor can be determined.For example, an affinity label can be fused to either the amino- orcarboxyl-terminus of the ligand. Such label may be a FLAG epitope tag,or, e.g., an Ig or Fc domain. An expression library can be screened forspecific binding of the cytokine, e.g., by cell sorting, or otherscreening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho et al., (1993) Proc. Natl. Acad. Sci. USA90:11267–11271; and Liu et al., (1994) J. Immunol. 152:1821–29.Alternatively, a panning method may be used. See, e.g., Seed and Aruffo,(1987) Proc. Natl. Acad. Sci. USA 84:3365–3369.

Protein cross-linking techniques with label can be applied to isolatebinding partners of the p40/IL-B30 cytokine complex. This would allowidentification of proteins which specifically interact with thecytokine, e.g., in a ligand-receptor like manner.

Early experiments will be performed to determine whether the known IL-6or G-CSF receptor components are involved in response(s) to p40/IL-B30.It is also quite possible that these functional receptor complexes mayshare many or all components with a p40/IL-B30 receptor complex, eithera specific receptor subunit or an accessory receptor subunit.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES

I. General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis et al., (1982) Molecular Cloning, A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook et al.,(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1–3, CSHPress, NY; Ausubel et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; Ausubel et al., (1987 and Supplements) Current Protocolsin Molecular Biology Wiley/Greene, NY; Innis et al., (eds. 1990) PCRProtocols: A Guide to Methods and Applications Academic Press, NY;Bonifacino et al., Current Protocols in Cell Biology Wiley, NY; andDoyle et al., Cell and Tissue Culture: Laboratory Protocols Wiley, NY.Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel et al.,(1987 and periodic supplements); Deutscher, (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan et al., (1995 and supplements) Current Protocols inProtein Science John Wiley and Sons, New York, N.Y.; Matsudaira, (ed.1993) A Practical Guide to Protein and Peptide Purification forMicrosequencing, Academic Press, San Diego, Calif.; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments (epitopetags), e.g., to a FLAG sequence or an equivalent which can be fused,e.g., via a protease-removable sequence. See, e.g., Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87–98, PlenumPress, NY; and Crowe et al., (1992) QIAexpress: The High LevelExpression & Protein Purification System QUIAGEN, Inc., Chatsworth,Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the University of Wisconsin GeneticsComputer Group (GCG), Madison, Wis., the NCBI at NIH, and GenBank, NCBI,EMBO, and other sources of public sequence. Other analysis sourcesinclude, e.g., RASMOL program, see Bazan et al., (1996) Nature 379:591;Lodi et al., (1994) Science 263:1762–1766; Sayle and Milner-White,(1995) TIBS 20:374–376; and Gronenberg et al., (1991) ProteinEngineering 4:263–269; and DSC, see King and Sternberg, (1996) ProteinSci. 5:2298–2310. See, also, Wilkins et al., (eds. 1997) ProteomeResearch: New Frontiers in Functional Genomics Springer-Verlag, NY;Salzberg et al., (eds. 1998) Computational Methods in Molecular BiologyElsevier, NY; and Birren et al., (eds. 1997) Genome Analysis: ALaboratory Manual Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

Standard immunological techniques are described, e.g., in Hertzenberg etal., (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1–4,Blackwell Science; Coligan, (1991 and updates) Current Protocols inImmunology Wiley/Greene, NY; and Methods in Enzymology vols. 70, 73, 74,84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. Cytokine assays aredescribed, e.g., in Thomson, (ed. 1994) The Cytokine Handbook (2d ed.)Academic Press, San Diego; Metcalf and Nicola, (1995) The HematopoieticColony Stimulating Factors Cambridge University Press; and Aggarwal andGutterman, (1991) Human Cytokines Blackwell Pub.

Assays for vascular biological activities are well known in the art.They will cover angiogenic and angiostatic activities in tumor, or othertissues, e.g., arterial smooth muscle proliferation (see, e.g., Koyomaet al., (1996) Cell 87:1069–1078), monocyte adhesion to vascularepithelium (see McEvoy et al., (1997) J. Exp. Med. 185:2069–2077), etc.See also Ross, (1993) Nature 362:801–809; Rekhter and Gordon, (1995) Am.J. Pathol. 147:668–677; Thyberg et al., (1990) Atherosclerosis10:966–990; and Gumbiner, (1996) Cell 84:345–357.

Assays for neural cell biological activities are described, e.g., inWouterlood, (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

FACS analyses are described in Melamed et al., (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro, (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson et al., (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Cloning of Human p40 and/or IL-B30

The IL-12 p40 sequences are available from various sequence databases,as described above. The sequence of the IL-B30 gene is provided inTable 1. The sequence is derived from a genomic human sequence.

These sequences allow preparation of PCR primers, or probes, todetermine cellular distribution of the genes. The sequences allowisolation of genomic DNA which encode the messages.

Using the probe or PCR primers, various tissues or cell types are probedto determine cellular distribution. PCR products are cloned using, e.g.,a TA cloning kit (Invitrogen). The resulting cDNA plasmids are sequencedfrom both termini on an automated sequencer (Applied Biosystems).

III. Cellular Expression of p40 and IL-B30

An appropriate probe or primers specific for cDNA encoding therespective genes are prepared. Typically, the probe is labeled, e.g., byrandom priming. Coordinate expression of both subunits is most importantwhere the p40/IL-B30 complex is of interest.

IV. Purification of p40/IL-B30 Protein

Multiple transfected cell lines are screened for one which expresses thecytokine or complex at a high level compared with other cells.Alternatively, a combination recombinant construct can be made. Variouscell lines are screened and selected for their favorable properties inhandling. Individual isolation of the respective subunits andcombination thereafter may result in some dimer formation. NaturalIL-B30 can be isolated from natural sources, or by expression from atransformed cell using an appropriate expression vector. Adenovirusconstructs can also be used for production/expression.

Purification of the expressed subunits or complex is achieved bystandard procedures, or may be combined with engineered means foreffective purification at high efficiency from cell lysates orsupernatants. In particular, fusion of p40 to IL-B30, with or withoutappropriate linker, can result in high efficiency methods for processingor purification. FLAG or His₆ segments can be used for such purificationfeatures. Alternatively, affinity chromatography may be used withspecific antibodies, see below.

Protein is produced in coli, insect cell, or mammalian expressionsystems, as desired.

V. Preparation of Antibodies Specific for p40/IL-B30

Synthetic peptides or purified protein are presented to an immune systemto generate monoclonal or polyclonal antibodies. See, e.g., Coligan,(1991) Current Protocols in Immunology Wiley/Greene; and Harlow andLane, (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.Immunoselection or depletion methods can be applied to ensure thatresulting antibodies are specific for antigenic determinants presentedby the complex of polypeptides, distinct from those presented by theindividual components themselves. Polyclonal serum, or hybridomas may beprepared. In appropriate situations, the binding reagent is eitherlabeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods. Immunoselection,immunodepletion, and related techniques are available to prepareselective reagents, as desired, e.g., for the complex between the twosubunits.

VI. IL-12 p40 and IL-B30 Coprecipitate

A mouse IL-12 p40-Ig fusion construct was prepared in an expressionvector. The Ig domain binds to Protein A, and can precipitate thatpolypeptide. An IL-B30Etag (epitope tagged with a FLAG motif at the Nterminus) construct was also prepared, which polypeptide isimmunoprecipitable with the M2 antibody. The expression constructs weretransfected into 293 T cells, either with the IL-12 p40-Ig constructalone, the IL-B30Etag construct alone, or both together. Cells werelabeled with ³⁵S methionine. With the IL-12 p40 construct alone, nosoluble protein was detected in the cell supernatant using Protein A.Likewise, with the

FLAG-IL-B30 construct, no soluble protein was detected in the cellsupernatant using the M2 antibody. However, with cotransfection of thetwo expression constructs, the cell supernatant produced a solublecomplex which was precipitable with either the Protein A reagent or theM2 antibody. PAGE analysis of the complex revealed that the Protein Aprecipitated complex was made of polypeptides corresponding to the twoexpected polypeptides IL-12 p40-Ig fusion and the FLAG-IL-B30polypeptides. Correspondingly, the complex precipitated with the M2antibody was made up of the FLAG-IL-B30 polypeptide and the IL-12 p40-Igfusion protein.

Similar experiments with a human IL-12 p40 expression construct and ahuman FLAG-IL-B30 construct provided the expected results. Transfectionwith the FLAG-IL-B30 construct resulted in no significant solubleprotein. Cotransfection of both expression vectors into primate cellsresulted in effective secretion of a complex which wasimmunoprecipitable with the M2 antibody. PAGE analysis of the resultingcomplex confirmed that the complex was made up of the FLAG-IL-B30polypeptide and the IL-12 p40 polypeptide.

VII. Receptor Identification

The IL-12 receptor is made up of the IL-12 receptor subunits β1 and β2.A fusion construct of p40/IL-B30 binds to cells expressing the receptorsubunit β1.

A homodimer of the IL-12 p40 subunits can block the binding of IL-12 tothe mouse subunit β1, but not to the subunit β2. The p40 subunit is acomponent of the p40/IL-B30 complex, so it was tested whether the IL-12receptor subunit β1 could be a component of the receptor for a fusionconstruct of p40/IL-B30. Antibodies to the IL-12 receptor subunit β1block binding of the fusion construct to cells expressing the receptorsubunit β1. Antibodies against the p40/p70 complex, mainly recognizingthe p40 subunit, can block the effect of the p40/IL-B30 composition,suggesting that the p40 component is important in receptor interaction.These observations suggest that the receptor subunit β1 binds to thep40/IL-B30 fusion construct. Similar experiments testing involvement ofthe common gp130 subunit shared among related receptors suggest that thegp130 is not a relevant subunit of the receptor for p40/IL-B30.

Having identified one subunit of the receptor, expression cloningefforts have been initiated. Cells expressing this one subunit butshowing no binding will be used to expression clone an additionalsubunit. Other receptor subunit β2 homologs are being screened.Alternatively, libraries from appropriate cells can be used in standardexpression cloning methods.

VIII. Evaluation of Breadth of Biological Functions

Biological activities of p40/IL-B30 complex are tested based, e.g., onthe sequence and structural homology between IL-B30 and IL-6 and G-CSF.Initially, assays that had shown biological activities of IL-6 or G-CSFwere examined. Assays were performed on either recombinant complex orfusion construct. Fusion construct consisted of a construct with theIL-12 p40 signal sequence linked to an N terminal FLAG epitope fused tothe mature IL-12 p40 sequence fused to a ser/gly rich linker sequence ofappropriate length fused to the mature sequence of IL-B30. Thisconstruct both expresses well, is secreted, and the epitope tag allowsboth purification and localization. Both mouse and human sequence formswere generated. Adenovirus expression constructs of both separatepolypeptides and fusion proteins are also made available.

Target cell types include lympoid, myeloid, mast, pre-B, pre-T, andfibroblast-endothelial cell types. For example, macrophage/monocytecells will be evaluated for cell surface marker changes, e.g., MHC classII, B7, CD40, and related families; cytokine and chemokine production;and antigen presentation capacity. CD4+ T cells, both naive CD45Rb^(hi)and memory CD45Rb^(low) T cells, will be assayed, e.g., for growth andactivation markers, and for effector functions, e.g., cytokine andchemokine production. Cytotoxic CD4+, CD8+ and NK cells will beevaluated for effects on generation and function. Effects on antibodyproduction will be tested, e.g., on splenic and MLN B cells. Dendriticcells will be evaluated for generation, maturation, and function,including factor production. Apoptosis assays are also being developed.

Long term bone marrow cultures will be tested for effects on modulationof stroma cells and stem cell generation and differentiation (Dextercultures), for modulation of stromal cells and B cell progenitorgeneration and differentiation (Whitlock-Witte cultures), and forevaluation of potential to regulate primitive myeloid and B lymphoidpopulations.

A. Effects on Proliferation of Cells

The effect on proliferation of various cell types are evaluated withvarious concentrations of cytokine. A dose response analysis isperformed, in combinations with the related cytokines IL-6, G-CSF, etc.A cytosensor machine may be used, which detects cell metabolism andgrowth (Molecular Devices, Sunnyvale, Calif.).

Human p40/IL-B30 fusion protein enhanced proliferation of human PHAblasts stimulated with anti-CD3 or both anti-CD3 and anti-CD28. Theanti-CD3 stimulation appears to be essential. Human p40/IL-B30 fusionprotein also enhanced proliferation of activated Th1 or Th2 cell clones,but not resting Th1 or Th2 cell clones.

Either mouse or human fusion protein worked on mouse target cells.Fusion protein supported proliferation of CD4+ CD45Rb^(low) CD62L^(low)CD44^(hi) cells (memory/activated T cells) when stimulated withanti-CD3. Stimulation by fusion protein is not enhanced by anti-CD28costimulation. This is not grossly dependent on presence of IL-2. Thissuggests that p40/IL-B30 may be an important factor for expanding apopulation of cells with a memory phenotype and/or generating ormaintaining immunologic memory. This cytokine seems to selectivelysupport activated memory cells with a Th1 phenotype, e.g., cells whichproduce IFNγ, but no IL-4 or IL-5.

B. Effects on Differentiation of Naive T Cells

Human cord blood cells were collected and naive CD4+ T cells wereisolated. These were cultured, e.g., for 2 weeks, in the presence ofanti-CD3 and IL-2 and with irradiated fibroblasts expressing CD32, CD58,and CD80, thereby activating and proliferating T cells. The T cellculture was evaluated for effects of various cytokines on proliferationor differentiation. Individual cells were evaluated for cytokineproduction by FACS analysis. The p40/IL-B30 fusion protein supported theproliferation and differentiation of T cells producing IFNγ and no IL-4,a cytokine expression profile characteristic of Th1 cells.

C. Effects on the Expression of Cell Surface Molecules

Monocytes are purified by negative selection from peripheral bloodmononuclear cells of normal healthy donors. Briefly, 3×10⁸ ficoll bandedmononuclear cells are incubated on ice with a cocktail of monoclonalantibodies (Becton-Dickinson; Mountain View, Calif.) consisting, e.g.,of 200 μl of αCD2 (Leu-5A), 200 μl of αCD3 (Leu-4), 100 μl of αCD8 (Leu2a), 100 μl of αCD19 (Leu-12), 100 μl of αCD20 (Leu-16), 100 μl of αCD56(Leu-19) , 100 μl of αCD67 (IOM 67; Immunotech, Westbrook, Me.), andanti-glycophorin antibody (10F7MN, ATCC, Rockville, Md.). Antibody boundcells are washed and then incubated with sheep anti-mouse IgG coupledmagnetic beads (Dynal, Oslo, Norway) at a bead to cell ratio of 20:1.Antibody bound cells are separated from monocytes by application of amagnetic field. Subsequently, human monocytes are cultured in Yssel'smedium (Gemini Bioproducts, Calabasas, Calif.) containing 1% human ABserum in the absence or presence of IL-B30, IL-6, G-CSF or combinations.

Analyses of the expression of cell surface molecules can be performed bydirect immunofluorescence. For example, 2×10⁵ purified human monocytesare incubated in phosphate buffered saline (PBS) containing 1% humanserum on ice for 20 minutes. Cells are pelleted at 200×g. Cells areresuspended in 20 ml PE or FITC labeled mAb. Following an additional 20minute incubation on ice, cells are washed in PBS containing 1% humanserum followed by two washes in PBS alone. Cells are fixed in PBScontaining 1% paraformaldehyde and analyzed on FACScan flow cytometer(Becton Dickinson; Mountain View, Calif.). Exemplary mAbs are used,e.g.: CD11b (anti-mac1), CD11c (a gp150/95), CD14 (Leu-M3), CD54 (Leu54), CD80 (anti-BB 1/B7), HLA-DR (L243) from Becton-Dickinson and CD86(FUN 1; Pharmingen), CD64 (32.2; Medarex), CD40 (mAb89; Schering-PloughFrance).

D. Effects on Cytokine Production by Human Cells

Human monocytes are isolated as described and cultured in Yssel's medium(Gemini Bioproducts, Calabasas, Calif.) containing 1% human AB serum inthe absence or presence of IL-B30 (1/100 dilution baculovirus expressedmaterial). In addition, monocytes are stimulated with LPS (E. coli0127:B8 Difco) in the absence or presence of IL-B30 and theconcentration of cytokines (IL-1β, IL-6, TNFα, GM-CSF, and IL-10) in thecell culture supernatant determined by ELISA.

For intracytoplasmic staining for cytokines, monocytes are cultured (1million/ml) in Yssel's medium in the absence or presence of IL-B30 andLPS (E. coli 0127:B8 Difco) and 10 mg/ml Brefeldin A (Epicentretechnologies Madison Wis.) for 12 hrs. Cells are washed in PBS andincubated in 2% formaldehyde/PBS solution for 20 minutes at RT.Subsequently cells are washed, resuspended in permeabilization buffer(0.5% saponin (Sigma) in PBS/BSA (0.5%)/Azide (1 mM)) and incubated for20 minutes at RT. Cells (2×10⁵) are centrifuged and resuspended in 20 mldirectly conjugated anti-cytokine mAbs diluted 1:10 in permeabilizationbuffer for 20 minutes at RT. The following antibodies can be used:IL-1α-PE (364-3B3-14); IL-6-PE (MQ2-13A5); TNFα-PE (MAb11); GM-CSF-PE(BVD2-21C11); and IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.).Subsequently, cells are washed twice in permeabilization buffer and oncein PBS/BSA/Azide and analyzed on FACScan flow cytometer (BectonDickinson; Mountain View, Calif.).

Human PHA blasts produced IFNγ in response to contacting with humanp40/IL-B30 fusion construct. The effects were synergistic with IL-2.Fusion product enhanced IFNγ production by activated, but not resting Tcells, resting Th1 cell clones, or resting Th2 cell clones.

E. Effects on Proliferation of Human Peripheral Blood Mononuclear Cells(PBMC)

Total PBMC are isolated from buffy coats of normal healthy donors bycentrifugation through ficoll-hypaque as described (Boyum et al.). PBMCare cultured in 200 μl Yssel's medium (Gemini Bioproducts, Calabasas,Calif.) containing 1% human AB serum in 96 well plates (Falcon,Becton-Dickinson, NJ) in the absence or presence of IL-B30. Cells arecultured in medium alone or in combination with 100 U/ml IL-2 (R&DSystems) for 120 hours. 3H-Thymidine (0.1 mCi) is added during the lastsix hours of culture and 3H-Thymidine incorporation determined by liquidscintillation counting.

The native, recombinant, and fusion proteins would be tested for agonistand antagonist activity in many other biological assay systems, e.g., onT-cells, B-cells, NK, macrophages, dendritic cells, hematopoieticprogenitors, etc. Because of the IL-6 and G-CSF structural relationship,assays related to those activities should be analyzed

p40/IL-B30 is evaluated for agonist or antagonist activity ontransfected cells expressing IL-6 or G-CSF receptor and controls. See,e.g., Ho et al., (1993) Proc. Natl. Acad. Sci. USA 90, 11267–11271; Hoet al., (1995) Mol. Cell. Biol. 15:5043–5053; and Liu et al., (1994). J.Immunol. 152:1821–1829.

p40/IL-B30 is evaluated for effect in macrophage/dendritic cellactivation and antigen presentation assays, T cell cytokine productionand proliferation in response to antigen or allogenic stimulus. See,e.g., de Waal Malefyt et al., (1991) J. Exp. Med. 174:1209–1220; de WaalMalefyt et al., (1991) J. Exp. Med. 174:915–924; Fiorentino et al.,(1991) J. Immunol. 147, 3815–3822; Fiorentino et al., (1991) J. Immunol.146:3444–3451; and Groux et al., (1996) J. Exp. Med. 184:19–29.

p40/IL-B30 will also be evaluated for effects on NK cell stimulation.Assays may be based, e.g., on Hsu et al., (1992) Internat. Immunol.4:563–569; and Schwarz et al., (1994) J. Immunother. 16:95–104.

B cell growth and differentiation effects will be analyzed, e.g., by themethodology described, e.g., in Defrance et al., (1992). J. Exp. Med.175:671–682; Rousset et al., (1992) Proc. Natl. Acad. Sci. USA89:1890–1893; including IgG2 and IgA2 switch factor assays.

IX. Generation and Analysis of Genetically Altered Animals

Transgenic mice can be generated by standard methods. Such animals areuseful to determine the effects of overexpression of the genes, inspecific tissues, or completely throughout the organism. Such mayprovide interesting insight into development of the animal or particulartissues in various stages. Moreover, the effect on various responses tobiological stress can be evaluated. See, e.g., Hogan et al., (1995)Manipulating the Mouse Embryo: A Laboratory Manual (2d ed.) Cold SpringHarbor Laboratory Press.

Adenovirus techniques are available for expression of the genes invarious cells and organs. See, e.g., Hitt et al., (1997) Adv. Pharmacol.40:137–195; and literature from Quantum Biotechnologies, Montreal,Canada. Animals may be useful to determine the effects of the genes onvarious developmental or physiologically functional animal systems.

A 0.5 kb cDNA encoding for IL-B30 was cloned as an EcoRI fragment intoan expression vector containing the CMV enhancer β-actin promoter andthe rabbit β-globin polyadenylation signal, previously described by Niwaet al., (1991) Gene 108:193–200. Separation of the transgene from vectorsequence was accomplished by zonal sucrose gradient centrifugation asdescribed by Mann et al., (1993) “Factor Influencing ProductionFrequency of Transgenic Mice”, Methods in Enzymology 225: 771–781.Fractions containing the transgene were pooled, microcentrifugationthrough Microcon-100 filters and washed 5 times with microinjectionbuffer (5 mM Tris-HCl, pH 7.4, 5 mM NaCl, 0.1 mM EDTA).

The transgene was resuspended in microinjection buffer (5 mM Tris-HCl,pH 7.4, 5 mM NaCl, 0.1 mM EDTA) to a final concentration of 1–5 ng/ml,microinjected into ([C57BL/6J×DBA/2]F₁; The Jackson Laboratory) eggs,which were then transferred into oviducts of ICR (Sprague-Dawley) fostermothers, according to published procedures by Hogan et al., (1994)Manipulation of the Mouse Embryo, Plainview, N.Y., Cold Spring HarborLaboratory Press. By 10 days of life, a piece of tail from the resultinganimals was clipped for DNA analysis. Identification of transgenicfounders was carried out by polymerase chain reaction (PCR) analysis, aspreviously described by Lira et al., (1990) Proc. Natl. Acad. Sci. USA,87: 7215–7219. Identification of the IL-B30 transgenic mice wasaccomplished by amplification of mouse tail DNA. As an internal controlfor the amplification reaction primers for the endogenous LDL gene wereused. The primers amplify a 200 bp segment of the IL-B30 transgene and397 bp segment of the LDL gene. PCR conditions were: 95° C., 30 seconds;60° C., 30 seconds; 72° C., 60 seconds for 30 cycles. Transgenic animalswere kept under pathogen-free conditions.

Analysis of IL-B30 Transgenic Mice

RNA was extracted from tissues using RNA STAT-60, followingspecifications from the manufacturer (TEL-TEST, Inc. Friendswood, Tex.).Total RNA (20 mg) was denatured and blotted onto Biotrans membrane (ICNBiomedicals, Costa Mesa, Calif.). Transgene expression was assessed byhybridization to randomly labeled L-30 cDNA (Stratagene, La Jolla,Calif.). Acute phase liver gene expression was assessed by hybridizingtotal RNA with randomly labeled PCR segments of the murine hemopexingene, of the murine alpha-1-acid glycoprotein, and of the murinehaptoglobin gene.

ELISA kits were purchased from commercial sources and run according tothe manufacturer's instructions. ELISA kits for murine IL-2(sensitivity<3 pg/ml), murine IL-1b (sensitivity<3 pg/ml), murineIFN-gamma (sensitivity<2 pg/ml) and murine TNF-alpha (sensitivity<5.1pg/ml) were purchased from R & D systems (Minneapolis, Minn.). MurineIL-6 ELISA kits (sensitivity<8 pg/ml) were purchased from BiosourceInternational (Camarillo, Calif.). Murine IL-1α ELISA kits(sensitivity<6 pg/ml) were purchased from Endogen (Cambridge, Mass.).

ELISA assays for serum immunoglobulin levels were run using antibodypairs purchased from PharMingen (San Diego, Calif.) following themanufacturer's guidelines. Anti-mouse IgM (clone 11/41), anti-mouse IgA(clone R5-140), anti-mouse IgG1 (clone A85-3), anti-mouse IgG2a (cloneR11-89) and anti-mouse IgG2b (clone R9-91) were used as captureantibodies. Purified mouse IgM (clone G155-228), IgA (clone M18-254),IgG1 (clone 107.3), IgG2a (clone G155-178) and IgG2b (clone 49.2) wereused to generate standard curves. Biotin anti-mouse IgM (clone R6-60.2),biotin anti-mouse IgA (clone R5-140), biotin anti-mouse IgG1 (cloneA85-1), biotin anti-mouse IgG2a (clone R19-15) and biotin anti-mouseIgG2b (clone R12-3) were used as detection antibodies.

Levels of IGF-1 in mouse serum were determined using a commerciallyavailable radioimmunoassay for human IGF-1 that also recognizes murineIGF-1 after serum samples were acid-ethanol extracted according toinstructions provided by the manufacturer (Nichols Institute, San JuanCapistrano, Calif.).

After sacrifice, tissues were either snap frozen with freezing media forcryosection, or fixed by immersion in 10% phosphate-buffered formalin.Formalin fixed tissues were routinely processed at 5 mm, and werestained with hematoxylin and eosin (H & E). For immunostaining, snapfrozen sections were fixed with acetone and air dried.

Blood samples were collected from the infra-orbital sinus into sterile,evacuated tubes with added EDTA (Vacutainer Systems; Becton Dickinson,Rutherford, N.J.). Hematologic values were determined with an automatedsystem (Abbot Cell-Dyn 3500, Abbot Park, Ill.). Platelet counts wereperformed manually when the instrument was unable to provide accurateplatelet counts due to excessive clumping or excessively largeplatelets. Blood smears were stained with Modified Wright-Giemsa stain(Hema-Tek Stain Pack, Bayer Corp., Elkhart, Ind.) using an automatedstainer (Bayer Hema-Tek 2000, Elkhart, Ind.) and examined manually forimmature cells and platelet, red blood cell, and white blood cellmorphology.

Bone Marrow Transfer

The femur and tibia were cleaned of muscle and bone marrow was expelledby flushing the bone with PBS. Bone marrow cells were washed once, andinjected i.v. into recipient mice which had been lethally irradiated(1000 RAD).

Phenotype of IL-B30 Transgenic Mice

To analyze the biological function of IL-B30, the gene was expressedunder the control of the CMV enhancer/actin promoter, described by Niwaet al., (1991) Gene 108:193–200, in transgenic mice. Thisenhancer/promoter cassette directs high levels of transgene expressionprimarily to skeletal muscle and pancreas, but the transgene can beexpressed in virtually all organs and cells. See Lira et al., (1990)Proc. Natl. Acad. Sci. USA 87: 7215–7219.

IL-B30 transgenic mice were runted compared to control littermates. Therate of body weight gain in IL-B30 transgenic mice varied widely but wasclearly lower than that found in control littermates. Northern blotanalysis, of RNA extracted from either muscle or skin of IL-B30transgenic mice and control littermates hybridized to IL-B30 cDNA,revealed that IL-B30 mRNA was detected in both muscle and skin of allIL-B30 transgenic mice, whereas no IL-B30 mRNA was detected in controllittermates. This demonstrated that stunted growth was always associatedwith expression of IL-B30.

Of the IL-B30 transgenic mice obtained, 25% survived to adulthood andwere affected by expression of IL-B30 as exhibited by impaired growth, aswollen abdomen, ruffled fur, infertility and sudden death. Thus,transgenic expression of IL-B30 caused a phenotype that prevented thegeneration of IL-B30 transgenic progeny. The results presented here arederived from the preliminary analysis of IL-B30 transgenic founder mice.

Histological Analysis of IL-B30 Transgenic Mice

Microscopical examination of tissues collected from IL-B30 transgenicmice revealed minimal to moderate inflammation in multiple sites,including the lung, skin, esophagus, small intestine and liver (bileducts), large intestine, and pancreas. Inflammatory infiltratesconsisted of neutrophils, lymphocytes, and/or macrophages. Inflammationin the skin was associated with acanthosis and/or ulceration in somemice. In the lungs, peribronchial mononuclear cell infiltrates weresometimes prominent, alveolar walls contained increased numbers ofleukocytes, and the epithelium lining airways was hyperplastic. Minimalperiportal mononuclear cell infiltrates were also common in the liver.The cortex of lymph nodes was sometimes sparsely cellular and lackedfollicular development.

Extramedullary hematopoiesis (EMH) was observed in the liver, spleen,and lymph nodes. The EMH was especially marked in the spleen. Thespleens from three transgenic mice and one control mouse were examinedafter immunohistological staining for T cells (anti-CD3), B cells(anti-B220), and macrophages (anti-F4/80). In the transgenic mice theCD3-, B220-, and F4/80-positive cells were present in their normallocations. However, the white pulp was separated by the EMH in the redpulp, and the positively staining cells within the red pulp wereinterspersed with hematopoietic cells that stained with less intensity,or did not stain positively, with the various antibodies. Theseobservations demonstrate that transgenic expression of IL-B30 inducessystemic inflammation that is associated with EMH.

To analyze the effect of IL-B30 on leukocyte and platelet counts in theperipheral blood, a complete blood analysis was performed. The number ofneutrophils in the blood of IL-B30 transgenic mice was increased 3- to11-fold over the highest neutrophil count in control littermates.Increases in peripheral blood neutrophils are typical of inflammationand correlate with the infiltration of neutrophils observed in varioustissues. Accordingly, the myeloid (granulocytic)/erythroid ratio wasincreased in the bone marrow.

In addition, the number of circulating platelets was increased up to3-fold in IL-B30 transgenic mice over control littermates. An increasednumber of platelets could originate from either an increased number ofmegakaryocytes, or from an increase in production of platelets bymegakaryocytes. To test either possibility, the peripheral blood, bonemarrow and spleen from IL-B30 transgenic mice were analyzedmicroscopically. In the peripheral blood, platelets of bizarremorphology, including elongated and spindle-shaped platelets, werefrequently detected. In bone marrow and spleen of some mice,megakaryocytes were enlarged due to increased amounts of cytoplasm. Incontrast, the number of megakaryocytes in bone marrow and spleen was notincreased. This suggests that IL-B30 induces an increase in the numberof platelets by accelerating their production by megakaryocytes.

All IL-B30 transgenic mice examined also suffered from mild to moderatemicrocytic hypochromic anemia with schistocytes and varying degrees ofregeneration evident. The hematocrit values were lower than the controlmean by 36 to 70%. The presence of microcytic hypochromic anemiasuggests a defect in hemoglobin production.

Cytokine Profile of IL-B30 Transgenic Mice

To test whether the systemic inflammation seen in IL-B30 transgenic micecorrelated with altered expression of pro-inflammatory cytokines, wedetermined the concentrations of IL-1, TNFα, IL-6, and IFNγ in theperipheral blood. In all IL-B30 transgenic mice tested, the levels ofTNFα and IFNγ were increased. In addition, the level of IL-1 wasincreased in 25% of IL-B30 transgenic mice tested. Concentrations ofIL-1 and TNFα found in IL-B30 transgenic mice reached levels associatedwith the induction of an acute inflammatory response by LPS.Surprisingly, no IL-6 was detected in the peripheral blood of IL-B30transgenic mice, even though expression of IL-6 is highly induced underinflammatory conditions (Reinecker et al., (1993) Clin. Exp. Immunology94: 174–181; Stevens et al., (1992) Dig. Dis. Sci. 37: 818–826) and canbe induced directly by TNFα, IL-1 and IFNγ (Helle et al., (1988) Eur. J.Immunol. 18: 957–959.

Acute Phase Genes in the Livers of IL-B30 Transgenic Mice

The body reacts to inflammation with an acute phase responsecharacterized by the expression of defined plasma proteins in the liver.Since IL-B30 transgenic mice exhibit a phenotype characteristic ofsystemic inflammation, we examined the expression of acute phase genesin the livers of IL-B30 transgenic mice and control littermates. Theacute phase liver genes alpha-1-acid glycoprotein, haptoglobin andhemopexin were highly expressed in all IL-B30 transgenic mice tested,while no expression of these genes was detected in control littermates.This demonstrates that acute phase liver genes are constitutivelyexpressed in IL-B30 transgenic animals.

Serum Immunoglobulins of IL-B30 Transgenic Mice

During an immune response, some cytokines induce B cell differentiationand subsequent immunoglobulin synthesis. To test whether immunoglobulinsynthesis was altered in IL-B30 transgenic mice, the concentrations ofimmunoglobulin isotypes in the peripheral blood were determined. In 2 of7 IL-B30 transgenic mice, the concentration of IgA was increased 6- to9-fold when compared to control littermates. Furthermore, theconcentrations of IgG1, IgG2a and IgG2b were increased 2.5 to 6-fold inall IL-B30 transgenic mice tested when compared to control littermates.In contrast, no significant increase in IgM or IgE titers could bedetected in any of the IL-B30 transgenic mice tested. In fact, 4 of 7IL-B30 transgenic mice displayed markedly decreased levels of IgMsynthesis. In summary, a subset of IL-B30 transgenic mice displayed a 6-to 9-fold increase in the concentrations of immunoglobulin isotypes IgAand IgG, whereas no significant increase was detected in theconcentrations of immunoglobulin isotypes IgM and IgE.

Serum IGF-1 Levels in IL-B30 Transgenic Mice

Chronic inflammatory conditions (Kirschner and Sutton, (1986)Gastroenterology 91: 830–836; Laursen et al., (1995) Arch. Dis. Child.72:494–497) or overexpression of cytokines in transgenic animals (DeBenedetti et al., (1994) J. Clin. Invest. 93: 2114–2119) can causegrowth impairment that is associated with a decrease of insulin-likegrowth factor-1 (IGF-1). To test whether stunted growth of IL-B30transgenic mice could be traced to reduced levels of IGF-1, serumsamples of transgenic mice were assayed for IGF-1. In all IL-B30transgenic mice tested, the amount of IGF-1 in the serum was 12 to 14%of the level found in age-matched control littermates. This suggeststhat transgenic expression of IL-B30, as well as the subsequentinflammatory response produced, results in the reduction of IGF-1 inIL-B30 transgenic mice, and could consequently be the cause of impairedgrowth and infertility (Gay et al., (1997) Endocrinology 137(7):2937–2947).

Expression of Biologically Active IL-B30 in Hematopoietic Cells

Cytokines are secreted proteins that regulate the immune system locallyor mediate long-range effects. To test whether IL-B30 functions as acytokine and can induce distant multi-organ inflammation and an acutephase liver response, we transferred IL-B30 transgenic bone marrow intolethally irradiated wildtype recipient mice.

Bone marrow recipients were monitored weekly for the induction of anacute phase response. Increased concentrations of the acute phaseprotein SAA could be detected in IL-B30 bone marrow recipients as earlyas 35 days post transfer and levels of SAA increased over time.Concurrent with increasing concentrations of SAA in the peripheral bloodthe health of IL-B30 bone marrow recipients deteriorated as judged bythe appearance of ruffled fur and inflamed skin around the snout andthroat. In contrast, recipients of wildtype bone marrow did not haveelevated levels of SAA in blood, nor did they appear sick.

Animals were terminated when they appeared severely sick. Expression ofIL-B30 could be detected in the bone marrow and spleen of recipients ofIL-B30 transgenic bone marrow, but not in organs of recipients ofwildtype bone marrow. As in IL-B30 transgenic donors skin, lung, liver,and the gastrointestinal tract were inflamed in recipients of IL-B30transgenic bone marrow, but not in wildtype bone marrow recipients.Again acute phase liver genes (hemopexin, AGP-1) were highly expressedin IL-B30 transgenic bone marrow recipients, but no IL-6 could bedetected in blood serum. These results suggest that IL-B30 is a truecytokine with long-ranging properties.

Transgenic expression of IL-B30 induces a striking phenotypecharacterized by runting, systemic inflammation, infertility and deathof transgenic animals. IL-B30 transgenic animals have systemicinflammation with infiltration of inflammatory cells into lung, liver,skin, and the digestive tract.

Overexpression of IL-B30 in vivo caused a phenotype of impaired growthand inflammation—that was strikingly similar to that of several modelsof transgenic expression of IL-6. Similar to the effect of transgenicexpression of IL-6, or after administration of recombinant IL-6 to mice,neutrophil infiltration and anemia were observed in animals as a resultof transgenic expression of IL-B30. As in IL-6 transgenic animals,impaired growth of IL-B30 transgenic founders was linked to decreasedlevels of IGF-1 that might be related to the systemic inflammationobserved in these animals.

This phenotype of IL-B30 transgenic animals could be caused byupregulated IL-6 expression either as a direct effect of IL-B30overexpression, or by IL-B30 mediated upregulation of IL-1 and TNFαexpression. IL-1 and TNFα are known inducers of IL-6 and increasedconcentrations of TNFα and IL-1 were found in the peripheral blood ofIL-B30 transgenic mice.

However, no IL-6 could be detected in blood of IL-B30 transgenicsuggesting that the phenotype of IL-B30 animals is directly linked tooverexpression of this novel cytokine and, as had been implied by theirsequence homologies, that IL-B30 has biological activities similar toIL-6.

IL-6 is a pleiotropic cytokine that among a wide variety of functionsinduces thrombocytosis, acute-phase protein synthesis, and B celldifferentiation.

Indeed, IL-B30 transgenic animals express constitutively acute phaseliver genes like AGP-1, haptoglobin, hemopexin and serum amyloid Aprotein. A similar phenotype has been shown in mice as an effect oftransgenic overexpression of IL-6, or after administration ofrecombinant IL-6. In addition, transgenic expression of IL-B30 resultedin thrombocytosis that was unusual in that many of the platelets hadbizarre morphology (elongated appearance, large size, and/or spindleshapes). We suspect that IL-B30 and/or other upregulated cytokines havean effect on normal platelet production. This suggests again that IL-B30shares a biological activity with IL-6 and its relatives.

IL-6 has also been identified as a B cell differentiation factor.Transgenic overexpression of IL-6 causes plasmocytoma and IL-6 deficientmice show a reduced IgG response. While we saw increases in IgG and IgAproduction in some IL-B30 transgenic mice, this observation was notconsistent between different founders. Thus further analysis is neededto characterize IL-B30 further as a potential B cell differentiationfactor.

In IL-B30 transgenic mice increases in circulating neutrophils wereconsistent with the inflammation evident in various tissues, however,the changes in red blood cell parameters are not as easily explained.IL-1, TNF-alpha, and IFN-gamma are mediators of a syndrome commonlycalled anemia of chronic disease (ACD), which generally presents as anormocytic, normochromic, nonregenerative (or minimally regenerative)anemia and is seen in a variety of chronic inflammatory diseases. Anemiaof Chronic Disease may also present as microcytic, hypochromic in somehuman patients. The syndrome is due to altered iron metabolism anddiminished response to erythropoietin. The microcytic hypochromic anemiaobserved in the IL-B30 mice may be due to ACD, as suggested by theincreases in peripheral cytokine concentrations. However, the mostcommon cause of microcytic hypochromic anemia is iron deficiency, whichis more consistent with the partial bone marrow response (regeneration)and thrombocytosis seen in the IL-B30 mice. Further investigation,including measurement of serum ferritin, iron, and total iron bindingcapacity, which would allow differentiation of ACD from iron deficiencyanemia, was not undertaken due to the difficulties in obtaining adequateblood from affected mice.

IL-1 and TNF-alpha are known inducers of IL-6 expression and IL-6expression is usually upregulated during an inflammatory response.Therefore it is surprising that IL-6 could not be detected in theperipheral blood of IL-B30 transgenic animals. This suggests that IL-B30has a negative effect on IL-6 expression by a yet unidentifiedmechanism. Indeed the absence of IL-6 in IL-B30 transgenic animals mightexplain the high levels of IL-1 and TNF-alpha observed in these animalssince IL-6 has a negative effect on the concentrations of circulatingIL-1 and TNF-alpha in mice. The high concentrations of circulatingTNF-alpha observed in IL-B30 transgenic mice could also be an result ofthe increased concentrations of IFN-gamma. IFN-gamma is produced by IL-2activated T cells or IL-4-activated B cells, and induces the expressionof TNF-alpha in monocytes and macrophages. It remains to be determinedwhether expression of IFN-gamma is mediated directly by IL-B30 or byother cytokines induced by IL-B30. In summary, our results suggest thatIL-B30 shares a wide variety of biological activities with IL-6. Itremains to be seen whether these biological activities are mediated by acommon receptor, a signal transduction element or transcription factorshared with IL-6. These issues will hopefully be clarified by ongoingexperiments using genetic and biochemical approaches.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A composition comprising a substantially pure polypeptide comprisingan IL-12 p40 fused to an IL-B30, wherein the IL-12 p40 is encoded by themature coding sequence of SEQ ID NO: 19, and the IL-B30 is encoded bythe mature coding sequence of SEQ ID NO:1.
 2. The composition of claim 1wherein the polypeptide is a fusion protein comprising, from theN-terminus to the C-terminus, the IL-12 p40, a linker, and the IL-B30.3. A nucleic acid encoding a fusion protein comprising an IL-12 p40fused to an IL-B30, wherein the IL-12 p40 is encoded by the maturecoding sequence of SEQ ID NO: 19, and the IL-B30 is encoded by themature coding sequence of SEQ ID NO:1.
 4. The nucleic acid of claim 3wherein the fusion protein comprises, from the N-terminus to theC-terminus, the IL-12 p40, a linker, and the IL-B30.
 5. The nucleic acidof claim 4 prepared by using synthetic DNA encoding the IL-12 p40, thelinker, and the IL-B30.
 6. A method of preparing a fusion protein ofIL-12 p40 and IL-B30, said method comprising: (1) providing anexpression vector that comprises the nucleic acid of claim 3; (2)expressing the fusion protein in a cell using said expression vector;and (3) preparing the fusion protein from said cell.
 7. The method ofclaim 6 wherein the fusion protein comprises, from the N-terminus to theC-terminus, the IL-12 p40, a linker, and the IL-B30.
 8. An isolatednucleic acid encoding a fusion protein comprising an IL-12 p40 fused toan IL-B30, wherein the IL-12 p40 is encoded by a sequence that is atleast 95% identical to SEQ ID NO:19, the IL-B30 is encoded by a sequencethat is at least 95% identical to SEQ ID NO:1, and the fusion proteinenhances or inhibits production of IFNγ, TNFα and/or IL-1.
 9. Anisolated cell comprising the nucleic acid of claim
 7. 10. A compositioncomprising a polypeptide encoded by the nucleic acid of claim
 8. 11. Thecomposition of claim 10 wherein the polypeptide is a fusion proteincomprising, from the N-terminus to the C-terminus, the IL-12 p40, alinker, and the IL-B30.
 12. The composition of claim 10, furthercomprising an acceptable carrier.
 13. The cell of claim 9, which is aninsect cell.
 14. A method of preparing a fusion protein of IL-12 p40 andIL-B30, said method comprising: (1) providing an expression vector thatcomprises the nucleic acid of claim 8; (2) expressing the fusion proteinin a cell using said expression vector; and (3) preparing the fusionprotein from said cell.
 15. The method of claim 14 wherein the fusionprotein comprises, from the N-terminus to the C-terminus, the IL-12 p40,a linker, and the IL-B30.
 16. The method of claim 15 wherein theexpression vector is prepared by using synthetic DNA encoding the IL-12p40, the linker, and the IL-B30.
 17. The nucleic acid of claim 8 furthercomprising a signal sequence.
 18. The method of claim 14 wherein theexpression vector further comprises a signal sequence.
 19. An isolatednucleic acid encoding a fusion protein comprising an IL-12 p40 fused toan IL-B30, wherein the IL-12 p40 is encoded by a sequence that is atleast 95% identical to SEQ ID NO:19, the IL-B30 is encoded by a sequencethat is at least 95% identical to SEQ ID NO:1, and the fusion proteinstimulates or inhibits proliferation of memory T cells.
 20. Acomposition comprising a substantially pure polypeptide encoded by thenucleic acid of claim
 19. 21. A method of preparing a fusion protein ofIL-12 p40 and IL B30, said method comprising: (1) providing anexpression vector that comprises the nucleic acid of claim 19; (2)expressing the fusion protein in a cell using said expression vector;and (3) preparing the fusion protein from said cell.
 22. A compositioncomprising a polypeptide encoded by the nucleic acid of claim 19.